Anna Globinska

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients

Background: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. Objective: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. Methods: The expression, regulation, and function of TJs were determined in air‐liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. Results: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL‐4 and IL‐13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens‐1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL‐4 and IL‐13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. Conclusion: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL‐4, and IL‐13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.

MicroRNAs and the immune response to respiratory virus infections

MicroRNAs (miRNAs) are small ssRNA molecules, which are involved in gene expression regulation at the post-transcriptional level. Their biological functions include modulation of both innate and adaptive immune response. miRNAs participate in the maintenance of the airway epithelial barrier and are also implicated in the modulation of antiviral defense in epithelial cells. The immune response to respiratory viruses such as rhinovirus, influenza virus and respiratory syncytial virus is associated with an altered expression of distinct miRNAs, and the changes in the miRNA expression profile in epithelial cells may contribute to the pathogenesis of both acute and chronic airway disease. Understanding the role of these small molecules in the antiviral immune response and identification of miRNAs target genes may help to clarify the mechanisms of virus-host interaction, and in the future may lead to development of new antiviral treatments.

Novel mechanisms in immune tolerance to allergens during natural allergen exposure and allergen-specific immunotherapy

Allergen-specific immunotherapy (AIT) has been used for more than 100 years as a clinical tolerance-inducing and immune tolerance-inducing therapy for allergic diseases and represents a potentially curative method of treatment. AIT functions through multiple mechanisms including early desensitization of basophils and mast cells, regulating T-cell and B-cell responses, changing antibody isotypes, and decreasing activation, mediator release and affected tissue migration of eosinophils, basophils, and mast cells. Similar molecular and cellular mechanisms have been observed in subcutaneous AIT, sublingual AIT and peptide immunotherapy as well as natural tolerance to high doses of allergen exposure in beekeepers and cat owners.

Impaired virus replication and decreased innate immune responses to viral infections in nasal epithelial cells from patients with allergic rhinitis.

The aim of this study was to assess the immune response to parainfluenza virus type 3 (PIV3), rhinovirus 1B (RV1B) and intracellular Toll‐like receptors (TLR) agonists in nasal epithelial cells (NECs) from patients with allergic rhinitis and healthy controls. NECs were obtained from eight patients with allergic rhinitis (AR) and 11 non‐atopic healthy controls (HC) by nasal scraping, grown to confluence and exposed to PIV3, RV1B infection or TLR‐3 and TLR‐7/8 agonists. Interferon (IFN)‐λ1, IFN‐α, IFN‐β and regulated on activation, normal T expressed and secreted (RANTES) release into the cell culture supernatants was assessed at 8, 24 and 48 h upon infection or 8 and 24 h after stimulation with poly(I:C) and R848. mRNA levels of IFNs, RANTES, interferon regulatory transcription factor (IRF)3, IRF7 and viral gene copy number were determined using real‐time polymerase chain reaction (RT‐PCR). PIV3 but not RV1B replication 48 h after infection was significantly lower (P < 0·01) in NECs from AR patients compared to HC. PIV3 infection induced significantly less IFN‐λ1 (both protein and mRNA) in NECs from AR compared to HC. IFN‐β mRNA expression and RANTES protein release and mRNA expression tended to be smaller in AR compared HC cells in response to both viruses. Stimulation with TLR‐3 agonist [poly (I:C)] induced similar IFN‐λ1 and RANTES generation in AR and HC subjects. Viral infections in NECs induced IRF7 expression, which correlated with IFN and RANTES expression. These data suggest that virus proliferation rates and the immune response profile are different in nasal epithelial cells from patients with allergic rhinitis compared to healthy individuals.

Modified Allergens for Immunotherapy

Purpose of ReviewDuring the past few decades, modified allergens have been developed for use in allergen-specific immunotherapy (AIT) with the aim to improve efficacy and reduce adverse effects. This review aims to provide an overview of the different types of modified allergens, their mechanism of action and their potential for improving AIT.Recent FindingsIn-depth research in the field of allergen modifications as well as the advance of recombinant DNA technology have paved the way for improved diagnosis and research on human allergic diseases. A wide range of structurally modified allergens has been generated including allergen peptides, chemically altered allergoids, adjuvant-coupled allergens, and nanoparticle-based allergy vaccines. These modified allergens show promise for the development of AIT regimens with improved safety and long-term efficacy. Certain modifications ensure reduced IgE reactivity and retained T cell reactivity, which facilities induction of immune tolerance to the allergen. To date, multiple clinical trials have been performed using modified allergens. Promising results were obtained for the modified cat, grass and birch pollen, and house dust mite allergens.SummaryThe use of modified allergens holds promise for improving AIT efficacy and safety. There is however a need for larger clinical studies to reliably assess the added benefit for the patient of using modified allergens for AIT.

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients

Purpose In order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls. Methods HBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR. Results PIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics. Conclusions The immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.

Respiratory syncytial virus infection influences tight junction integrity

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air–liquid interface (ALI) and infected with RSV and ultraviolet (UV)‐irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV‐irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post‐infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down‐regulated claudin‐1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin‐1 and claudin‐7 at protein level.

The lack of L-PG production and the repercussions of it in regards to M. Tuberculosis interactions with mononuclear phagocytes

The lysine connection with phosphatidylglycerol (PG) alters the M. tuberculosis(Mtb) surface charge, and consequently it may decrease the bacterial vulnerability to antimicrobial action of the immune cells. The aim of the study was to assess the significance of PG lysinylation in the Mtb interactions with mononuclear phagocytes. Both the Mtb strain with deletion of lysX gene (Mtb-lysX) which is responsible for PG lysinylation as well as the complemented strain (Mtb-compl) was used to infect human blood monocytes or THP-1 cells. The monocytes were obtained by MACS technique, or THP-1 cells. The Mtb-lysX strain has exhibited the enhanced sensitivity to HNP 1-3. However, it was not susceptible to bactericidal action of cathepsin G. The LysX deletion did not influence the Mtb ability of monocyte induction to IL-10 secretion. The intra- and extracellular expression of MHC-II was similarly reduced after the Mtb-lysX or Mtb-Rv infections. Noticeably significant is that the Mtb strain with deleted lysX has not affected the intensity of the gene expression of cathepsin G compared to the uninfected monocytes. That is the clear contrast to what the Mtb-Rv strain has proved. The obtained results suggest that the Mtb ability to lysinylate PG is a participatory element in mycobacterial strategy of survival inside phagocytic cells. However, the extended studies are needed to determine its influence on the other immune cells and define its role in the developing of Mtb infection.

Innate lymphoid cells: the role in respiratory infections and lung tissue damage

ABSTRACT Introduction: Innate lymphoid cells (ILCs) represent a diverse family of cells of the innate immune system, which play an important role in regulation of tissue homeostasis, immunity and inflammation. Emerging evidence has highlighted the importance of ILCs in both protective immunity to respiratory infections and their pathological roles in the lungs. Therefore, the aim of this review is to summarize the current knowledge, interpret and integrate it into broader perspective, enabling greater insight into the role of ILCs in respiratory diseases. Areas covered: In this review we highlighted the role of ILCs in the lungs, citing the most recent studies in this area. PubMed searches (2004- July 2017) were conducted using the term ‘innate lymphoid cells respiratory viral infections’ in combination with other relevant terms including various respiratory viruses. Expert commentary: Since studies of ILCs have opened new areas of investigation, understanding the role of ILCs in respiratory infections may help to clarify the mechanisms underlying viral-induced exacerbations of lung diseases, providing the basis for novel therapeutic strategies. Potential therapeutic targets have already been identified. So far, the most promising strategy is cytokine-targeting, although further clinical trials are needed to verify its effectiveness.

Mechanisms of allergen-specific immunotherapy: Diverse mechanisms of immune tolerance to allergens

OBJECTIVE The aim of this review is to provide an overview of the current knowledge on the mechanisms of allergen immunotherapy based on the recent publications and clinical trials. DATA SOURCES PubMed literature review. STUDY SELECTIONS In this review, we focus on diverse mechanisms of AIT and provide an insight into alternative routes of administration. Additionally, we review and discuss the most recent studies investigating potential biomarkers and highlight their role in clinical settings. RESULTS Successful allergen-specific immunotherapy (AIT) induces the reinstatement of tolerance toward allergens and represents a disease-modifying treatment. In the last decades, substantial progress in understanding the mechanisms of AIT has been achieved. Establishment of long-term clinical tolerance to allergens engages a complex network of interactions, modulating the functions of basophils, mast cells, allergen-specific regulatory T and B cells, and production of specific antibodies. The reduction of symptoms and clinical improvement is achieved by skewing the immune response away from allergic inflammation. CONCLUSION Although the complex nature of AIT mechanisms is becoming more clear, the need to discover reliable biomarkers to define patients likely to respond to the treatment is emerging.