Role of airway epithelial cell miRNAs in asthma

The airway epithelial cells and overlying layer of mucus are the first point of contact for particles entering the lung. The severity of environmental contributions to pulmonary disease initiation, progression, and exacerbation is largely determined by engagement with the airway epithelium. Despite the cellular cross-talk and cargo exchange in the microenvironment, epithelial cells produce miRNAs associated with the regulation of airway features in asthma. In line with this, there is evidence indicating miRNA alterations related to their multifunctional regulation of asthma features in the conducting airways. In this review, we discuss the cellular components and functions of the airway epithelium in asthma, miRNAs derived from epithelial cells in disease pathogenesis, and the cellular exchange of miRNA-bearing cargo in the airways

Sepsis-Like Systemic Inflammation Induced by Nano-Sized Extracellular Vesicles From Feces

Nano-sized extracellular vesicles (EVs), including exosomes, microvesicles, and other types of vesicles, are released by most mammalian cells and bacteria. We here ask whether feces contain EVs of mammalian and/or bacterial origin, and whether these EVs induce systemic inflammation. Fecal extracellular vesicles (fEVs) were isolated from mice and humans. The presence of EVs from Gram-negative and Gram-positive bacteria was detected by enzyme-linked immunosorbent assay using anti-lipid A and anti-lipoteichoic acid antibodies, whereas Western blot using anti-beta-actin antibody was employed to detect host-derived EVs in the fEVs. Further, fEVs were administered into mice by intraperitoneal injection, and inflammatory responses were investigated in the peritoneum, blood, and lungs. The role of TLR2 and TLR4 were studied using knockout mice. Significant quantities of EVs were present in feces from mice as well as humans, and derived from Gram-negative and Gram-positive bacteria, as well as the host. Bacteria-free fEVs introduced into the peritoneum induced local and systemic inflammation (including in the lungs), but fEVs from germ-free animals had weaker effects. This pronounced local and systemic inflammatory responses seemed to be induced by EVs from both Gram-negative and Gram-positive bacteria, and was attenuated in mice lacking TLR2 or TLR4. Our findings show that fEVs cause sepsis-like systemic inflammation, when introduced intraperitoneally, a process regulated by TLR2 and TLR4.11Ysciescopu

Sensitization to molecular dog allergens in an adult population : Results from the West Sweden Asthma Study

Background: As the prevalence of dog allergy rises, component resolved diagnosis might improve the diagnosis, understanding of the clinical outcomes and the effectiveness of immunotherapy. Considering the paucity of data in adults, the current study characterized the patterns of sensitization to dog molecular allergens in an adult population. Methods: Data were derived from the West Sweden Asthma Study, a population-based and representative sample of adults from western Sweden. Of the 2006 subjects clinically examined, 313 participants sensitized to whole dog allergen extract were measured for specific immunoglobulin E (sIgE) levels to Can f 1, Can f 2, Can f 3, Can f 4, Can f 5 and Can f 6 using ImmunoCAP™. Polysensitization was defined as sensitization to ≥3 components. Overlapping sensitization was defined as having concomitant sensitization to at least two dog molecular allergen families (lipocalin, albumin or prostatic kallikrein). Results: Of 313, 218 (70%) subjects tested positive to at least one dog allergen component. Sensitization to Can f 1 (43%) was the most common, followed by Can f 5 (33%) among molecular allergens, while sensitization to lipocalins (56%) was the most common among component families. Polysensitization was found in 22% of all participants and was more common in participants with than in those without asthma. Subjects with asthma were less likely to be monosensitized to Can f 5 than those without asthma. Subjects with asthma had higher IgE levels of Can f 3, Can f 4 and Can f 6 than those without asthma. Overlapping sensitizations also differed between those with asthma and allergic rhinitis and those without. Conclusion: Increased knowledge about the sensitization patterns of dog allergen components can aid in defining their role in asthma and rhinitis. In complex clinical cases of dog allergy, a detailed analysis of dog allergen components can provide additional information on the nature of sensitization.publishedVersionPeer reviewe

LPA5 Is Abundantly Expressed by Human Mast Cells and Important for Lysophosphatidic Acid Induced MIP-1β Release

Background: Lysophosphatidic acid (LPA) is a bioactive lipid inducing proliferation, differentiation as well as cytokine release by mast cells through G-protein coupled receptors. Recently GPR92/LPA5 was identified as an LPA receptor highly expressed by cells of the immune system, which prompted us to investigate its presence and influence on mast cells. Principal Findings: Transcript analysis using quantitative real-time PCR revealed that LPA5 is the most prevalent LPA-receptor in human mast cells. Reduction of LPA5 levels using shRNA reduced calcium flux and abolished MIP-1β release in response to LPA. Conclusions: LPA5 is a bona fide LPA receptor on human mast cells responsible for the majority of LPA induced MIP-1β release

CpG methylation at GATA elements in the regulatory region of CCR3 positively correlates with CCR3 transcription

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription

Immunophenotyping of Circulating T Helper Cells Argues for Multiple Functions and Plasticity of T Cells In Vivo in Humans - Possible Role in Asthma

BACKGROUND: The immune process driving eosinophilic and non-eosinophilic asthma is likely driven by different subsets of T helper (Th) cells. Recently, in vitro studies and animal studies suggest that Th cell subsets displays plasticity by changing their transcription factor or by expressing multiple transcription factors. Our aim was to determine whether individuals with asthma and elevated circulating eosinophils express signs of different regulatory immune mechanisms compared with asthmatics with low blood eosinophils and non-asthmatic control subjects. In addition, determine the relationship between eosinophilia and circulating Th cell subsets. METHODOLOGY/PRINCIPAL FINDINGS: Participants were selected from a random epidemiological cohort, the West Sweden Asthma Study. Immunophenotypes of fresh peripheral blood cells obtained from stable asthmatics, with and without elevated eosinophilic inflammation (EOS high and EOS low respectively) and control subjects, were determined by flow cytometry. No differences in the number of Th1 (T-bet), Th2 (GATA-3), Th17 (RORγt) or Treg (FOXP3) cells were observed between the groups when analysing each subset separately. However, in all groups, each of the Th subsets showed expression of additional canonical transcription factors T-bet, GATA-3, RORγt and FOXP3. Furthermore, by in vitro stimulation with anti-CD3/anti-CD28 there was a significant increase of single expressing GATA-3(+) and co-expressing T-bet(+)GATA-3(+) cells in the EOS high asthmatics in comparison with control subjects. In addition, T-bet(-)GATA-3(+)RORγt(+)FOXP3(+) were decreased in comparison to the EOS low asthmatics. Finally, in a group of control subjects we found that the majority of proliferating Th cells (CD4(+)CD25(+)Ki67(+)) expressed three or four transcription factors. CONCLUSIONS: The ability of human Th cells to express several regulatory transcription factors suggests that these cells may display plasticity in vivo

Expansion of CD4+CD25+ and CD25- T-Bet, GATA-3, Foxp3 and RORγt Cells in Allergic Inflammation, Local Lung Distribution and Chemokine Gene Expression

Allergic asthma is associated with airway eosinophilia, which is regulated by different T-effector cells. T cells express transcription factors T-bet, GATA-3, RORγt and Foxp3, representing Th1, Th2, Th17 and Treg cells respectively. No study has directly determined the relative presence of each of these T cell subsets concomitantly in a model of allergic airway inflammation. In this study we determined the degree of expansion of these T cell subsets, in the lungs of allergen challenged mice. Cell proliferation was determined by incorporation of 5-bromo-2′-deoxyuridine (BrdU) together with 7-aminoactnomycin (7-AAD). The immunohistochemical localisation of T cells in the lung microenvironments was also quantified. Local expression of cytokines, chemokines and receptor genes was measured using real-time RT-PCR array analysis in tissue sections isolated by laser microdissection and pressure catapulting technology. Allergen exposure increased the numbers of T-bet+, GATA-3+, RORγt+ and Foxp3+ cells in CD4+CD25+ and CD4+CD25- T cells, with the greatest expansion of GATA-3+ cells. The majority of CD4+CD25+ T-bet+, GATA-3+, RORγt+ and Foxp3+ cells had incorporated BrdU and underwent proliferation during allergen exposure. Allergen exposure led to the accumulation of T-bet+, GATA-3+ and Foxp3+ cells in peribronchial and alveolar tissue, GATA-3+ and Foxp3+ cells in perivascular tissue, and RORγt+ cells in alveolar tissue. A total of 28 cytokines, chemokines and receptor genes were altered more than 3 fold upon allergen exposure, with expression of half of the genes claimed in all three microenvironments. Our study shows that allergen exposure affects all T effector cells in lung, with a dominant of Th2 cells, but with different local cell distribution, probably due to a distinguished local inflammatory milieu

Local and systemic mechanisms of allergen-induced airway inflammation

Allergic asthma is associated with pronounced inflammatory changes in the airways, including increased numbers of lymphocytes, neutrophils and mast cells, but most strikingly increased number of eosinophils. The accumulation of eosinophils within the airways in allergic airway inflammation is very likely a combination of increased production, migration and a prolonged survival of these cells. Eosinophils develop from CD34+ progenitor cells within the bone marrow (BM) and allergen challenge causes enhanced BM eosinophilopoiesis in asthmatic subjects as well as in animal models of allergen-induced airway inflammation. In addition, recent studies show that allergic subjects have increased number of CD34+ cells in BM and the airways.The aims of the present thesis were A) to determine which chemotactic factors govern the traffic of both the newly produced and the CD34+ eosinophils to the airways following allergen exposure, B) to establish whether or not the newly produced CD34+ eosinophilic cells have a capacity to proliferate locally within the airways following allergen exposure, and C) to evaluate the role of T lymphocytes in allergen-induced BM eosinophilopoiesis. In order to assess this, we used mouse models of allergen-induced airway inflammation. Wild type mice, IL-5 transgenic mice (NJ. 1638; CD3IL-5+) and mice deficient in CD4+ (CD4-/-) or CD8+ (CD8-/-) T cells were studied. Cells produced during the allergen exposure period were identified using a thymidine analogue, bromodeoxyuridine (BrdU). Local treatment with neutralizing anti-eotaxin-1 or anti-eotaxin-2 of allergen sensitized/exposed mice caused a significant reduction in newly produced and CD34+ BAL eosinophils. Significant airway concentrations of the neutralizing antibodies had to be achieved before a reduction in the number of allergen-induced airway eosinophils could be obtained. The expression of chemokine receptor 3 (CCR3) was upregulated on CD34+ BM cells leading to a significant increase of CD34+/CCR3+ eosinophil-lineage committed cells in BM, blood and BAL following allergen exposure. A fraction of the CD34+/CCR3+ cells proliferated in situ in response to allergen. In addition, in vitro colony formation of lung CD34+ cells was increased by IL-5 or eotaxin-2.Naïve crossbred CD3IL-5+/CD8-/- mice showed a significant reduction in the number of BM eosinophils when compared to naïve CD3IL-5+ or naïve crossbred CD3IL-5+/CD4-/- mice. Allergen exposed CD4-/- and CD8-/- mice had a significantly reduced number of BM and BAL eosinophils compared to wild type mice. Furthermore, a significant reduction in newly produced BM eosinophils (BrdU+/MBP+ cells) was found in both knockout strains when compared to allergen-challenged wild type mice. Eotaxin-2 levels in BALF of allergen-challenged CD4-/- mice were similar to saline exposed wild type mice.In conclusion, this thesis shows that the CCR3/eotaxin pathway is intricately involved in the regulation of allergen-driven in situ hematopoiesis and the accumulation/mobilization of CD34+ eosinophil-lineage committed cells in the lung. Allergen-induced BM eosinopilopoiesis is partly regulated by CD4+ and CD8+ T cells. In addition, CD4+ T cells may regulate IL-5 dependent eosinophil maturation in allergen-induced bone marrow eosinophilia. The subsequent traffic of eosinophils to the airways is likely to be at least partly regulated by CD4+ T cell dependent local airway eotaxin-2 production

INVESTIGATING INDUSTRY 4.0 READINESS IN THE SWEDISH MANUFACTURING SECTOR

Industry 4.0 is the fourth, most recent industrial revolution, and refers to the integration of the digital and the physical world in a manufacturing environment. Since first introduced in 2011, Industry 4.0 has gained significant academic and industrial attention. Due to the novelty of the concept, however, there are many areas that are yet to be properly covered in the Industry 4.0 literature. One area on which numerous calls for additional research have been made is Industry 4.0 readiness, which refers to the assessment of a company’s degree of readiness for a full-scale adoption of Industry 4.0 and its surrounding technologies. In order to respond to these calls for additional research, this study evaluates the Industry 4.0 readiness of a company in the Swedish manufacturing sector using a qualitative approach. The evaluation is based on a recently developed analytical framework which focuses on eight enabling technologies of Industry 4.0. In order to gain a more holistic understanding of the company’s Industry 4.0 readiness, a range of organizational barriers are also examined. The empirical findings reveal a varying degree of presence of the enabling technologies at the investigated company, consequently resulting in a degree of Industry 4.0 readiness of 63.2 %. An alternative degree of readiness is also calculated, taking into consideration the relative importance of the enabling technologies for the company. Finally, lack of an Industry 4.0 strategy, the existence of competency traps, limited financial support, and lack of internal collaborations are identified as the major organizational barriers to an increased Industry 4.0 readiness. By addressing these, it is argued that the company can facilitate their overall work with Industry 4.0 and thereby increase their readiness for a full-scale adoption of Industry 4.0