Julie Weidner

Background: Sex differences have been reported in the incidence, prevalence and severity of asthma. Previous findings from animal models have revealed sex‐related differences in inflammatory pathways that may contribute to asthma pathogenesis, but human studies are limited.Methods: Airway and blood samples (n = 55 and n = 85 respectively) were collected from adult females and males with asthma and healthy subjects. Type 2 innate lymphoid cells (ILC2s), T helper (Th)2 cells and their expression of IL‐33R/ST2 (ST2L) were evaluated by flow cytometry. IL‐13, thymic stromal lymphopoietin (TSLP), IL‐33 and soluble IL‐33R/ST2 (sST2) were measured by ELISA. Let‐7 miRNA expression in bronchial biopsies was determined by qPCR.Results: Females with asthma reported more exacerbations and had a higher number of airway eosinophils compared with males with asthma. Bronchial biopsy expression of Let‐7f, Let‐7g and miR‐98 tended to be higher in males with asthma compared with females and inversely correlated with asthma exacerbations. In contrast, increased levels of IL‐13, TSLP and sST2 were found in females with asthma compared with males.Conclusion: Our study demonstrates different inflammatory signatures between males and females with asthma. Let‐7 miRNAs act as immune modulators by inhibiting the production of IL‐13 and may be an important factor explaining the sex disparity seen in asthma.

MicroRNA (miRNA) mimics are an emerging class of oligonucleotide therapeutics, with a few compounds already in clinical stages. Synthetic miRNAs are able to restore downregulated levels of intrinsic miRNAs, allowing for parallel regulation of multiple genes involved in a particular disease. In this work, we examined the influence of chemical modifications patterns in miR-200c mimics, assessing the regulation of a selection of target messenger RNAs (mRNA) and, subsequently, of the whole transcriptome in A549 cells. We have probed 37 mimics and provided an initial set of instructions for designing miRNA mimics with potency and selectivity similar to an unmodified miRNA duplex. Additionally, we have examined the stability of selected mimics in serum. Finally, the selected two modification patterns were translated to two other miRNAs, miR-34a and miR-155. To differing degrees, these designs acted on target mRNAs in a similar manner to the unmodified mimic. Here, for the first time, we describe a structured overview of ‘miRNA mimics modification templates’ that are chemically stabilised and optimised for use in an in vitro set up and highlight the need of further sequence specific optimization when mimics are to be used beyond in vitro tool experiments.

The polarization of CD4+ T cells into different T helper subsets is an important process in many diseases, including asthma. Part of the adaptive immune system, T cells are responsible for propagating signals to alert and prime the immune system. MicroRNAs (miRNAs) are small non-coding RNAs that act on numerous targets in the cell to regulate a variety of cellular processes, including roles in T cell polarization. In this study, we aimed to identify genes dysregulated in peripheral blood mononuclear cells from individuals with asthma. Moreover, we sought to examine miRNAs that may regulate the candidate genes and explore their functional relationship. Utilizing a focused gene array, we identified the serum/glucocorticoid-regulated kinase 1 (SGK1) gene to be upregulated in circulating peripheral blood mononuclear cells, which included T cells, from individuals with asthma. Several miRNAs were bioinformatically identified to target SGK1, but miR-19a was the only screened candidate that negatively correlated to SGK1 expression. Further analysis of the miR-19a-SGK1 relationship showed a negative correlation in CD4+ T cells in situ and direct binding in vitro during T cell activation. Moreover, we observed a negative correlation of miR-19a and SGK1 during early type 2 polarization of CD4+ naïve human T cells. Thus, we suggest that miR-19a has a role in binding and regulating SGK1 transcript levels during T cell development.

Rationale: COPD has been associated repeatedly with single biomarkers of systemic inflammation, ignoring the complexity of inflammatory pathways. This study aimed to cluster patients with COPD based on systemic markers of inflammatory processes and to evaluate differences in their clinical characterisation and examine how these differences may relate to altered biological pathways.Methods: 213 patients with moderate-to-severe COPD in a clinically stable state were recruited and clinically characterised, which included a venous blood sample for analysis of serum biomarkers. Patients were clustered based on the overall similarity in systemic levels of 57 different biomarkers. To determine interactions among the regulated biomarkers, protein networks and biological pathways were examined for each patient cluster.Results: Four clusters were identified: two clusters with lower biomarker levels (I and II) and two clusters with higher biomarker levels (III and IV), with only a small number of biomarkers with similar trends in expression. Pathway analysis indicated that three of the four clusters were enriched in Rage (receptor for advanced glycation end-products) and Oncostatin M pathway components. Although the degree of airflow limitation was similar, the clinical characterisation of clusters ranged from 1) better functional capacity and health status and fewer comorbidities; 2) more underweight, osteoporosis and static hyperinflation; 3) more metabolically deranged; and 4) older subjects with worse functional capacity and higher comorbidity load.Conclusions: These new insights may help to understand the functionally relevant inflammatory interactions in the pathophysiology of COPD as a heterogeneous disease.

Single nucleotide polymorphisms (SNPs) in various genes have been shown to associate with COPD, suggesting a role in disease pathogenesis. Sulfatase modifying factor (SUMF1) is a key modifier in connective tissue remodelling, and we have shown previously that several SNPs inSUMF1are associated with COPD. The aim of this study was to investigate the association betweenSUMF1SNPs and advanced lung function characteristics.Never-, former and current smokers with (n=154) or without (n=405) COPD were genotyped for 21 SNPs inSUMF1and underwent spirometry, body plethysmography, diffusing capacity of the lung for carbon monoxide (D_LCO) measurement and impulse oscillometry.Four SNPs (rs793391, rs12634248, rs2819590 and rs304092) showed a significantly decreased odds ratio of having COPD when heterozygous for the variance allele, together with a lower forced expiratory volume in 1 s (FEV₁) and FEV₁/forced vital capacity (FVC) ratio and an impaired peripheral resistance and reactance. Moreover, individuals homozygous for the variance allele of rs3864051 exhibited a strong association to COPD, a lower FEV₁/FVC, FEV₁andD_LCO, and an impaired peripheral resistance and reactance. Other SNPs (rs4685744, rs2819562, rs2819561 and rs11915920) were instead associated with impaired lung volumes and exhibited a lower FVC, total lung capacity and alveolar volume, in individuals having the variance allele.Several SNPs in theSUMF1gene are shown to be associated with COPD and impaired lung function. These genetic variants ofSUMF1may cause a deficient sulfation balance in the extracellular matrix of the lung tissue, thereby contributing to the development of COPD.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post‐transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

Background: Asthma is a chronic inflammatory disease of the airways affecting more than 300 million people worldwide. The most common treatment for asthma is inhaled corticosteroids(ICS), but this treatment is not adequate for all individuals. microRNAs(miRs) were shown to be involved in asthma, allergy and inflammation, but their exact function in these processes remains largely unknown. In this study we asked whether ICS affected gene and miR expression in blood cells from allergic(AA) and non-allergic asthmatic(NAA) individuals. Methods: Healthy, AA and NAA individuals, which included ICS naïve and regular users, were recruited from the population based West Sweden Asthma Study. Peripheral blood mononuclear cells(PBMCs) were examined for alterations in gene and miR expression in the glucocorticoid signaling pathway. Results: We discovered that the healthy and asthma subgroups exhibited distinct changes on a genetic level, with subjects on ICS clustering more closely than ICS naïve. Interestingly, we found that in the asthma subgroups compared to healthy subjects, a transcription factor, POU2F1, and a kinase, SGK1, were down- and up-regulated, respectively. These alterations were most prominent in ICS naïve AAs. Additionally, miR-19a was predicted to target and appeared to correlate to the aforementioned genes suggesting involvement in the posttranscriptional regulation of glucocorticoid signaling. Conclusion: These findings suggest that alterations in glucocorticoid related genes and miR-19a may be influential in promoting asthma pathogenesis. Together these data will lead to a more thorough understanding of the complex regulation of gene and miR expression in asthma.

Background: Eosinophils develop from CD34⁺ progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation. Methods: Wild type (WT) and Rag1 deficient (Rag1^−/−) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1^−/− mice, lymphocyte deficient mice (Rag2^−/−Il2rg^−/−) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA. Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1^−/− mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1^−/− mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1^−/− and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2⁺ mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2^−/−Il2rg^−/− mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures. Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.

Type 2 innate lymphoid cells (ILC2s) and their adaptive counterpart type 2 T helper (T_H2) cells respond to interleukin-33 (IL-33) by producing IL-5, which is a crucial cytokine for eosinophil development in the bone marrow. The aim of this study was to determine if bone marrow ILC2s, T_H cells, and eosinophils are locally regulated by IL-33 in terms of number and activation upon exposure to the common aeroallergen house dust mite (HDM). Mice that were sensitized and challenged with HDM by intranasal exposures induced eosinophil development in the bone marrow with an initial increase of IL5Rα⁺ eosinophil progenitors, following elevated numbers of mature eosinophils and the induction of airway eosinophilia. Bone marrow ILC2s, T_H2, and eosinophils all responded to HDM challenge by increased IL-33 receptor (ST2) expression. However, only ILC2s, but not T_H cells, revealed increased ST2 expression at the onset of eosinophil development, which significantly correlated with the number of eosinophil progenitors. In summary, our findings suggest that airway allergen challenges with HDM activates IL-33-responsive ILC2s, T_H cells, and eosinophils locally in the bone marrow. Targeting the IL-33/ST2 axis in allergic diseases including asthma may be beneficial by decreasing eosinophil production in the bone marrow.

Background: Asthma is a chronic airway disease affecting millions of people. Better methods to define asthma subgroups using clinical parameters and molecular biomarkers are crucial in the development of personalized medicine. Objective: The aim of this study was to determine if circulating microRNAs (miRNAs) may be used to distinguish well–defined asthma groups. Methods: Blood serum from 116 well-defined subjects, including healthy controls and individuals with allergic or non-allergic asthma, from the West Sweden Asthma Study were included. Serum was analyzed for circulating miRNA expression of miR-126, − 145, −146a, − 155, − 223, and -374a and eosinophil cationic protein (ECP). Correlations between clinical characteristics and circulating miRNA expression as well as potential miRNA gene targets were investigated. Results: A subset of miRNAs were differentially expressed between allergic and non-allergic asthmatic individuals. Alterations in expression of miR-155, −146a, −374a and − 145 were observed in allergic asthmatics in response to inhaled corticosteroid usage. Additionally, miR-223 and miR-374a expression varied in non-allergic asthmatics based on blood eosinophil numbers. Numerous clinical parameters, including lung function measurements, correlated with subsets of miRNAs. Finally, pathway analysis revealed a potential role for inhaled corticosteroid induced miRNAs in leukocyte regulation, IL-6 signaling and glucocorticoid response. Conclusion: Circulating miRNA expression was altered in subjects with allergic and non-allergic asthma and correlated to clinical parameters including lung function and potential gene targets involved in immune processes. This combination of clinical and molecular data may be a basis for the further, more precise classification of asthma subgroups. Taken together, these findings would further asthma research and benefit future patients through the discovery of molecular mechanisms as well as identifying asthma subgroups contributing to the development of personalized medicine.