Multi-omics links IL-6 trans-signalling with neutrophil extracellular trap formation and Haemophilus infection in COPD.

BACKGROUND: Interleukin (IL)-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases; however, the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood. OBJECTIVE: Our aim was to identify and characterise COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation. METHODS: We used an IL-6TS-specific sputum biomarker profile (soluble IL-6 receptor (sIL-6R), IL-6, IL-1ß, IL-8, macrophage inflammatory protein-1ß) to stratify sputum data from patients with COPD (n=74; Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbation (BEAT-COPD)) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS by Haemophilus influenzae were studied in human neutrophils. RESULTS: Hierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, who shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterised by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (Chronic Respiratory Questionnaire total score; p=0.008), and increased levels of pro-inflammatory mediators and matrix metalloproteinases in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was identified as a potential mechanism for increased sIL-6R levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients. CONCLUSION: IL-6TS pathway activation due to chronic colonisation with Haemophilus may be an important disease driver in a subset of COPD patients.

A new house dust mite-driven and mast cell-activated model of asthma in the guinea pig.

BACKGROUND: Animal models are extensively used to study underlying mechanisms in asthma. Guinea pigs share anatomical, pharmacological and physiological features with human airways and may enable the development of a pre-clinical in vivo model that closely resembles asthma. OBJECTIVES: To develop an asthma model in guinea pigs using the allergen house dust mite (HDM). METHODS: Guinea pigs were intranasally sensitized to HDM which was followed by HDM challenges once weekly for five weeks. Antigen-induced bronchoconstriction (AIB) was evaluated as alterations in Rn (Newtonian resistance), G (tissue damping) and H (tissue elastance) at the first challenge with forced oscillation technique (FOT), and changes in respiratory pattern upon each HDM challenge were assessed as enhanced pause (Penh) using whole-body plethysmography. Airway responsiveness to methacholine was measured one day after the last challenge by FOT. Inflammatory cells and cytokines were quantified in bronchoalveolar lavage fluid, and HDM-specific immunoglobulins were measured in serum by ELISA. Airway pathology was evaluated by conventional histology. RESULTS: The first HDM challenge after the sensitization generated a marked increase in Rn and G, which was abolished by pharmacological inhibition of histamine, leukotrienes and prostanoids. Repeated weekly challenges of HDM caused increase of Penh and a marked increase in airway hyperresponsiveness for all three lung parameters (Rn , G and H) and eosinophilia. Levels of IgE, IgG1 , IgG2 and IL-13 were elevated in HDM-treated guinea pigs. HDM exposure induced infiltration of inflammatory cells into the airways with a pronounced increase of mast cells. Subepithelial collagen deposition, airway wall thickness and goblet cell hyperplasia were induced by repeated HDM challenge. CONCLUSION AND CLINICAL RELEVANCE: Repeated intranasal HDM administration induces mast cell activation and hyperplasia together with an asthma-like pathophysiology in guinea pigs. This model may be suitable for mechanistic investigations of asthma, including evaluation of the role of mast cells.

Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity.

Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.

Biological wound matrices with native dermis-like collagen efficiently modulate protease activity.

OBJECTIVE: When the delicate balance between catabolic and anabolic processes is disturbed for any reason, the healing process can stall, resulting in chronic wounds. In chronic wound pathophysiology, proteolytic imbalance is implicated due to elevated protease levels mediating tissue damage. Hence, it is important to design appropriate wound treatments able to control and modulate protease activity directly at the host/biomaterial interface. Here, we investigate collagen-based wound dressings with the focus on their potential to adsorb and inactivate tissue proteases. METHOD: We examined the effect of six collagen-based dressings on their ability to adsorb and inactivate different granulocyte proteases, plasmin, human neutrophil elastase (HLE), and matrix metalloproteases (MMP)-1, -2, -8, and -9, by an integrated approach including immunoelectron microscopy. RESULTS: We observed a reduction of the proteolytic activities of plasmin, HLE, and MMP-1, -2, -8, and -9, both on the biomaterial surface and in human chronic wound fluid. The most pronounced effect was observed in collagen-based dressings, with the highest content of native collagen networks resembling dermis structures. CONCLUSION: Our data suggest that this treatment strategy might be beneficial for the chronic wound environment, with the potential to promote improved wound healing.

Corrigendum: The pulmonary extracellular matrix is a bactericidal barrier against Haemophilus influenzae in chronic obstructive pulmonary disease (COPD): implications for an in vivo innate host defense function of collagen VI.

[This corrects the article DOI: 10.3389/fimmu.2018.01988.].

HDAC6 inhibitor ACY-1083 shows lung epithelial protective features in COPD.

Airway epithelial damage is a common feature in respiratory diseases such as COPD and has been suggested to drive inflammation and progression of disease. These features manifest as remodeling and destruction of lung epithelial characteristics including loss of small airways which contributes to chronic airway inflammation. Histone deacetylase 6 (HDAC6) has been shown to play a role in epithelial function and dysregulation, such as in cilia disassembly, epithelial to mesenchymal transition (EMT) and oxidative stress responses, and has been implicated in several diseases. We thus used ACY-1083, an inhibitor with high selectivity for HDAC6, and characterized its effects on epithelial function including epithelial disruption, cytokine production, remodeling, mucociliary clearance and cell characteristics. Primary lung epithelial air-liquid interface cultures from COPD patients were used and the impacts of TNF, TGF-ß, cigarette smoke and bacterial challenges on epithelial function in the presence and absence of ACY-1083 were tested. Each challenge increased the permeability of the epithelial barrier whilst ACY-1083 blocked this effect and even decreased permeability in the absence of challenge. TNF was also shown to increase production of cytokines and mucins, with ACY-1083 reducing the effect. We observed that COPD-relevant stimulations created damage to the epithelium as seen on immunohistochemistry sections and that treatment with ACY-1083 maintained an intact cell layer and preserved mucociliary function. Interestingly, there was no direct effect on ciliary beat frequency or tight junction proteins indicating other mechanisms for the protected epithelium. In summary, ACY-1083 shows protection of the respiratory epithelium during COPD-relevant challenges which indicates a future potential to restore epithelial structure and function to halt disease progression in clinical practice.

Bleomycin hydrolase regulates the release of chemokines important for inflammation and wound healing by keratinocytes.

Bleomycin hydrolase (BLMH) is a well-conserved cysteine protease widely expressed in several mammalian tissues. In skin, which contains high levels of BLMH, this protease is involved in the degradation of citrullinated filaggrin monomers into free amino acids important for skin hydration. Interestingly, the expression and activity of BLMH is reduced in patients with atopic dermatitis (AD) and psoriasis, and BLMH knockout mice acquire tail dermatitis. Apart from its already known function, we have discovered a novel role of BLMH in the regulation of inflammatory chemokines and wound healing. We show that lowered BLMH levels in keratinocytes result in increased release of the pro-inflammatory chemokines CXCL8 and GROα, which are upregulated in skin from AD patients compared to healthy individuals. Conditioned media from keratinocytes expressing low levels of BLMH increased chemotaxis by neutrophils and caused a delayed wound healing in the presence of low-level TNFα. This defective wound healing was improved by blocking the shared receptor of CXCL8 and GROα, namely CXCR2, using a specific receptor antagonist. Collectively, our results present a novel function of BLMH in regulating the secretion of chemokines involved in inflammation and wound healing in human keratinocytes.

The Pulmonary Extracellular Matrix Is a Bactericidal Barrier Against Haemophilus influenzae in Chronic Obstructive Pulmonary Disease (COPD): Implications for an in vivo Innate Host Defense Function of Collagen VI.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection in vivo. In vitro, collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and Haemophilus autotransporter protein (Hap). Similar observations were made in vivo specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.

Collagen VI Is Upregulated in COPD and Serves Both as an Adhesive Target and a Bactericidal Barrier for Moraxella catarrhalis.

Moraxella catarrhalis is a Gram-negative human mucosal commensal and pathogen. It is a common cause of exacerbation in chronic obstructive pulmonary disease (COPD). During the process of infection, host colonization correlates with recognition of host molecular patterns. Importantly, in COPD patients with compromised epithelial integrity the underlying extracellular matrix is exposed and provides potential adhesive targets. Collagen VI is a ubiquitous fibrillar component in the airway mucosa and has been attributed both adhesive and killing properties against Gram-positive bacteria. However, less is known regarding Gram-negative microorganisms. Therefore, in the present study, the interaction of M. catarrhalis with collagen VI was characterized. We found that collagen VI is upregulated in the airways of COPD patients and exposed upon epithelial desquamation. Ex vivo, we inoculated airway biopsies and fibroblasts from COPD patients with M. catarrhalis. The bacteria specifically adhered to collagen VI-containing matrix fibrils. In vitro, purified collagen VI microfibrils bound to bacterial surface structures. The primary adhesion target was mapped to the collagen VI α2-chain. Upon exposure to collagen VI, bacteria were killed by membrane destabilization in physiological conditions. These previously unknown properties of collagen VI provide novel insights into the extracellular matrix innate immunity by quickly entrapping and killing pathogen intruders.

Collagen VI encodes antimicrobial activity: novel innate host defense properties of the extracellular matrix.

Collagen type VI is a subepithelial extracellular matrix component in airways and an adhesive substrate for oral pathogens [Bober et al.: J Innate Immun 2010;2:160-166]. Here, we report that collagen VI displays a dose-dependent antimicrobial activity against group A, C, and G streptococci by membrane disruption in physiological conditions. The data disclose previously unrecognized aspects of the extracellular matrix in innate host defense.