An Alternate STAT6-Independent Pathway Promotes Eosinophil Influx into Blood during Allergic Airway Inflammation

Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4⁺ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues. We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4⁺ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4⁺ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4⁺ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation

Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells

There is great interest in substituting animal work with in vitro experimentation in human health risk assessment; however, there are only few comparisons of in vitro and in vivo biological responses to engineered nanomaterials. We used high-content genomics tools to compare in vivo pulmonary responses of multiwalled carbon nanotubes (MWCNT) to those in vitro in cultured lung epithelial cells (FE1) at the global transcriptomic level. Primary size, surface area and other properties of MWCNT- XNRI -7 (Mitsui7) were characterized using DLS, SEM and TEM. Mice were exposed via a single intratracheal instillation to 18, 54, or 162 μg of Mitsui7/mouse. FE1 cells were incubated with 12.5, 25 and 100 μg/ml of Mitsui7. Tissue and cell samples were collected at 24 hours post-exposure. DNA microarrays were employed to establish mechanistic differences and similarities between the two models. Microarray results were confirmed using gene-specific RT-qPCR. Bronchoalveolar lavage (BAL) fluid was assessed for indications of inflammation in vivo. A strong dose-dependent activation of acute phase and inflammation response was observed in mouse lungs reflective mainly of an inflammatory response as observed in BAL. In vitro, a wide variety of core cellular functions were affected including transcription, cell cycle, and cellular growth and proliferation. Oxidative stress, fibrosis and inflammation processes were altered in both models. Although there were similarities observed between the two models at the pathway-level, the specific genes altered under these pathways were different, suggesting that the underlying mechanisms of responses are different in cells in culture and the lung tissue. Our results suggest that careful consideration should be given in selecting relevant endpoints when substituting animal with in vitro testing

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil