This study determined the effect of BC on pollen-sensitized human nasal epithelial cells (hNECs) and showed that exposure to more than 50 µg/ml of BC or 25 g /ml BC + 200 g /ml pollen significantly decreased the viability of epithelial cells. Whilst exposure to 200 µg/ml BC, mainly up-regulated GO terms associated with hypoxia stress response, exposure to a combination of 25 µg/ml BC + 200 µg/ml pollen upregulated GO terms related to innate immune and inflammatory responses, including regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, degranulation and mast cells mediated immunity, and phagocytosis. Furthermore, increased expression of genes IL(interleukin)1B, CSF1 and FCER1G were found to be the main upstream regulators involved in the inflammatory process.
IL1B is a potent proinflammatory cytokine, belonging to the IL-1 family cluster that includes the IL-1a, and IL1-RN genes. IL-1β can be activated by caspase 1 and induces neutrophil influx and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and fibroblast proliferation and collagen production. IL1R1(interleukin-1 receptor 1) as a member of IL-1 family cluster may regulate the process of inflammation in organisms.11 One recent study has reported that exposure to BC induced differential expression of IL-1R1 in A549 cells,12 which is consistent with the findings from the present study.. Similarly, our findings for differential expression of other genes influencing neutrophils are consistent with other studies. For example, TGAM (integrin alpha M chain), a leukocyte-specific integrin, has been shown to be important in the regulation of neutrophil migration and phagocytosis-induced apoptosis in extravagated neutrophils.13 Similarly, Rac2, a member of the Rho family GTPases, which are critical in regulating neutrophil activation, is involved in the control of the neutrophil actin cytoskeleton, cell migration, and the NADPH oxidase.14 CSF3R (granulocyte colony-stimulating factor receptor) plays a crucial role in the proliferation, differentiation and survival of cells along the neutrophilic lineage. One of the most important functions of neutrophils is the production of oxidative metabolites for killing invading micro-organisms. Studies have suggested that S100A9 inhibits chemotaxis toward pro-inflammatory molecules and increased oxidative metabolism by neutrophils15, which is consistent with our finding of the anti-oxidative and anti-inflammatory effect of S100A9 on neutrophils. An in vitro study has confirmed that CCL20 has a direct chemotactic effect in neutrophil recruitment.16 In accordance with these studies, the present study has indicated that epithelium-derived neutrophil-activating peptide 78 (CXCL5), and neutrophil chemoattractants IL-8 (CXCL8) and CXCL3 are likely to be involved in the inflammatory response induced by BC and pollen in hNECs.
Macrophages are indispensable as members of the innate immune system, as they regulate normal physiology as first responders by communicating with the host’s adaptive immune system. Macrophage phenotypes include classically activated macrophages (M1) and alternatively activated macrophages (AAMφ or M2), of which M2 play a role in resolving inflammation. Canonical induction of M2 polarization is mediated by PPARs (peroxisome proliferator-activated receptors; PPARδ, PPARγ) and PGC1β (PPARγ coactivator-1 beta) activation, induction of cluster of differentiation 36 (CD36) and lipoprotein lipase (LPL). Several studies indicate that LPL is a primary regulator of macrophage lipid uptake and a modulator of macrophage polarity.17,18 Thus, the finding for increased expression of LPL in the present study would suggest that exposure to BC and pollen may induce macrophage polarization to M2. This is consistent with the finding for also increased expression of Colony-stimulating factor-1 (CSF-1, also known as macrophage-CSF); the primary regulator of the survival, proliferation, differentiation and function of mononuclear phagocytes; and CSF-1 receptor (CSF1R) following exposure to BC and pollen. Indeed, it has been demonstrated that CSF-1 enhances cytotoxicity, superoxide production, phagocytosis, chemotaxis and cytokine production in monocytes or macrophages when CSF-1 receptor (CSF1R) expressed on these cells is activated.19 Similarly, CSF-1 can also prime some innate immune responses while suppressing others by modulation of Toll-like receptors (TLRs), such as TLR2, another gene found to be differentially expressed following exposure to BC and pollen. Moreover, our findings for differential expression of MMP-9 and HCLS1 are also in accordance with studies that have demonstrated that MMP-9 activation is important for macrophage migration, and HCLS1 enhances the function of monocytes/ macrophages.20
Phagocytosis is primarily carried out by specialized cells termed professional phagocytes, which include cells of the immune system such as macrophages, neutrophils and dendritic cells. Of the differentially expressed genes associated with regulation of phagocytosis in the present study, pro-inflammatory cytokines IL1B (IL-1ß) and TNF-α, have been shown to upregulate Fc receptor-mediated phagocytosis.21 SLC11A1 (Solute Carrier Family 11, Member 1) was formerly known as natural resistance associated macrophage protein 1 (NRAMP), 22 and NRAMP1 is a member of the metal transporter protein family, which transfers iron (Fe) ions across the phagosome membrane.22 Integrin ITGSAM is important in the phagocytosis of complement coated particles, and may regulate phagocytosis-induced apoptosis in extravagated neutrophils. 23 DOCK2 is a new member of the CDM family proteins, which plays an important role in phagocytosis and NADPH oxidation by functioning upstream of RAC2.24 Protein Tyrosine Phosphatase Receptor Type C (PTPRC, CD45) has been shown to be involved in phagocytosis as a positive regulator of Src family kinases (SFKs). 25 CD36 and CD14 have been shown to interact with TLRs to induce phagocytosis and inflammation involving monocytes and macrophages. 26, 27 MERTK, a MER/AXL/TYRO3 receptor kinase family, plays a role in various processes such as macrophage clearance of apoptotic cells, platelet aggregation, cytoskeleton reorganization and engulfment. 28
The present study has demonstrated that exposure to BC and pollen also lead to differential expression of genes associated with regulation of mast cells. Mast cell mediator release plays a vital role in the initiation of inflammatory reactions associated with allergic disorders. This involves a chain of reactions following antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (FceRI) on the mast cell surface; involving activation of the Src family tyrosine kinase (Syk, include Lyn and Fgr), phosphorylation of the transmembrane adapter molecules linker for activated T cells 1 (LAT1) and LAT2, activation of protein kinase C (PKC) and liberation of intracellular calcium. These signals lead to mast cell degranulation and contribute to activation of transcription factors required for cytokine and chemokine production. The role of LAT2 in mast cell activation is still enigmatic; however, it has been proposed to both upregulate and downregulate antigen-mediated responses, and to enhance FceRI-dependent degranulation.29 In this pathway, tyrosine phosphorylation activates Vav1, a regulator of PLCγ-activated calcium signals to induce migration and activation of mast cells.30 FGR positively regulates mast cell degranulation, production of eicosanoids and cytokines,31 and FES protein-tyrosine kinase, a downstream effector of KIT signalling in mast cells, is required for migration of mast cells.