Carina Malmhäll

Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand.

Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells

Background: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34+ progenitor cells. Methods: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34+ progenitor cells. Results: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34+ hematopoietic progenitor cells. Conclusion: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34+ progenitor cells, implying a role in cells maturation process. To access the supplementary material to this article: Tables S1-S6, please see Supplementary files under Article Tools online.

MicroRNA-155 is essential for TH2-mediated allergen-induced eosinophilic inflammation in the lung

BACKGROUND Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored. OBJECTIVES We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation. METHODS To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin. RESULTS miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice. CONCLUSIONS Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1.

Effects of pollen and nasal glucocorticoid on FOXP3+, GATA-3+ and T-bet+ cells in allergic rhinitis.

Background:  T‐regulatory cells (Treg) affect the balance of TH2 and TH1 cells. Treg, TH2 and TH1 cells are regulated by the FOXP3, GATA‐3 and T‐bet transcription factors respectively. Our aim was to determine the number of FOXP3+, GATA‐3+ and T‐bet+ cells in nasal mucosa in symptom‐free allergic rhinitis (AR) patients vs healthy controls, as well as the effects of natural pollen exposure and concomitant nasal glucocorticoid treatment on these cells.

Increased number of CD34+ cells in nasal mucosa of allergic rhinitis patients: inhibition by a local corticosteroid

Background Eosinophils develop from CD34+ haematopoietic progenitor cells. Allergen exposure in susceptible individuals is known to induce a local eosinophilic inflammation, but the effect on progenitor cells is much less understood.

Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling.

BackgroundHuman cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.Methods and resultsExosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.ConclusionsOur results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

Interleukin-17-producing T-helper cells and related cytokines in human airways exposed to endotoxin

Previous studies on mouse models have indicated that interleukin (IL)-17 and IL-17-producing T-helper (Th) cells are important for pulmonary host defence against Gram-negative bacteria. Human correlates to these findings have not yet been demonstrated. The aim of the present study was to determine whether or not IL-17-producing Th cells are present and whether IL-17 and other Th17-associated cytokines are involved in the immunological response to endotoxin in human airways. Segmental exposure to endotoxin and contralateral exposure to vehicle were performed in the lungs of healthy volunteers, with subsequent bronchoalveolar lavage 12 or 24 h after exposure to study local changes in cytokines and inflammatory cells. Endotoxin exposure increased concentrations of IL-17, IL-22 and their downstream effector molecules, human &bgr;-defensin-2 and IL-8/CXC chemokine ligand 8, in bronchoalveolar lavage fluid. Th cells with the capacity to produce IL-17 were found among the bronchoalveolar lavage cells, and expression of IL-17 mRNA correlated with expression of the transcription factor, retinoic-acid-receptor-related orphan receptor C variant 2. Moreover, endotoxin increased the numbers of neutrophils, macrophages and IL-17-producing T-cells, as well as the concentration of the Th17-regulating cytokines, IL-21 and IL-23. In conclusion, IL-17-producing Th cells are present, and IL-17, as well as other Th17-associated cytokines, is involved in the immunological response to endotoxin in human airways.

MicroRNA-155 is a critical regulator of type 2 innate lymphoid cells and IL-33 signaling in experimental models of allergic airway inflammation

Background: Allergic airway inflammation is triggered by allergen exposure through several steps including release of IL‐33, which promotes cytokine (IL‐5, IL‐13) production by type 2 innate lymphoid cells (ILC2s). MicroRNA (miR)‐155 has recently been described to regulate adaptive responses in allergic inflammation. However, the role of miR‐155 in the regulation of ILC2s remains unexplored. Objective: We sought to elucidate the contribution of miR‐155 in ILC2 expansion using experimental murine models of allergic airway inflammation. Methods: To determine the role of miR‐155 in the regulation of ILC2s in allergic airway inflammation, miR‐155 deficient (miR‐155−/−) and wild‐type (WT) mice were subjected to acute or chronic allergen‐induced inflammation or treated with recombinant IL‐33. Results: miR‐155 was 10‐fold upregulated in WT‐derived ILC2s in response to IL‐33. Furthermore, miR‐155−/− mice demonstrated impaired lung IL‐33 levels in response to allergen challenge and the number of ILC2s was significantly reduced in allergen‐challenged miR‐155−/− mice compared with WT mice. Exogenous IL‐33 treatment revealed that miR‐155 is needed for IL‐33–induced ILC2 expansion and eosinophilic airway inflammation. Indeed, ILC2s from IL‐33–challenged miR‐155−/− lungs exhibited impaired proliferation, GATA‐3 expression, and IL‐13 production as compared with IL‐33–challenged WT ILC2s. Conclusions: Our findings for the first time demonstrate that ILC2s and IL‐33 signaling are regulated by miR‐155 in allergic airway inflammation.

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.

Local proliferation and mobilization of CCR3+ CD34+ eosinophil-lineage-committed cells in the lung

Emerging evidence suggests that haematopoietic CD34+ progenitor cells migrate from bone marrow (BM) to sites of allergen exposure where they can undergo further proliferation and final maturation, potentially augmenting the degree of tissue inflammation. In the current study we used a well‐characterized mouse model of allergen‐induced airway inflammation to determine the role of CCR3 receptor–ligand interactions in the migration and function of CD34+ cells. Allergen exposure significantly increased BM, blood and airway CD34+ CCR3+ cells as well as airway CD34+ CCR3+ stem cell antigen‐1‐positive (Sca‐1+) and CD34+  CD45+ interleukin‐5 receptor‐α‐positive (IL‐5Rα+) cells. A portion of the newly produced CD34+ CCR3+, Sca‐1+ CCR3+ and IL‐5Ralpha+ lung cells showed a significant proliferative capacity in response to allergen when compared with saline‐treated animals. In addition, in vitro colony formation of lung CD34+ cells was increased by IL‐5 or eotaxin‐2 whereas eotaxin‐2 had no effect on BM CD34+ cells. Furthermore, both eotaxin‐1 and eotaxin‐2 induced migration of BM and blood CD34+ CCR3+ cells in vitro. These data suggest that the CCR3/eotaxin pathway is involved in the regulation of allergen‐driven in situ haematopoiesis and the accumulation/mobilization of eosinophil‐lineage‐committed progenitor cells in the lung. Hence, targeting both IL‐5 and CCR3‐mediated signalling pathways may be required to control the inflammation associated with allergen‐induced asthma.