Assessment of Changes in Genetic Transcriptome in Nasal Epithelial Cells Exposed to Black Carbon and Pollen Allergen by High-throughput Transcriptomics


 Background: Evidence suggests that air pollution may be associated with an increase in airway responsiveness to allergens, an increase in bioavailability of airborne allergens and possibly exacerbation of allergic rhinitis (AR). Environmental black carbon (BC) is an important constituent of atmospheric particulate matter (PM), for which the mechanisms underlying its effects have not been fully elucidated in AR. The objective of the present study was to determine the BC and pollen-induced alterations in the transcriptome in human nasal epithelial cells (hNECs) in vitro.Methods: hNECs were prepared from nasal epithelial mucosal samples of healthy individuals undergoing nasal surgery (turbinoplasty or septoplasty). The hNECs were established as air-liquid interface (ALI) cultures and exposed to BC alone or in combination with pollen allergen. The changes in the transcriptome were analyzed by high-throughput RNA sequencing (RNA-Seq). Some of the differentially expressed genes were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Enrichment analysis based on Gene Ontology (GO) and KEGG database for each Gene was performed to determine their major biological functions and pathways.Results: Exposure to ≥50 μg/ml BC or 25μg/ml BC + 200g /ml pollen significantly decreased cell viability in the hNECs compared to control (p<0.05) or 25μg/ml BC alone (p<0.05). Expression of 114 genes (including 80 up-regulated and 34 down-regulated) and 293 genes (including 233 up-regulated and 60 down-regulated genes) was significantly altered following exposure to 200 μg/ml BC and 25 μg/ml BC =200 μg/ml pollen respectively. For 200 μg/ml BC, up-regulated GO terms were mainly associated with hypoxia stress response, whereas for 25 μg/ml BC + 200 μg/ml pollen treatment the top enriched GO terms were associated with inflammatory response including regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, and phagocytosis. KEGG pathway analysis indicated the top 10 upstream regulators to be IL1B, CSF1, CCL2, TLR2, LPL, IGF8, SPP1, CXCL8, FCER1G and IL1RN. The expressions of inflammation related gene IL1B, CSF1 and FCER1G were elevated as measured by RT-qPCR assay.Conclusion: BC and pollen allergen may induce innate immune and allergic inflammation in hNECs, and therefore potentially exacerbate the symptoms of AR in affected individuals.


Introduction
Allergic rhinitis (AR) is common in ammatory disease of the nasal mucosa, the prevalence of which has markedly increased over the past three decades and currently affects 10-40% of the population worldwide. 1 Epidemiologic studies have correlated the increase in AR in China with an increase in industrialization and air pollution over the last two decades. 2 Some studies have suggested that air pollution might induce an increase in airway responsiveness to allergens and the increase bioavailability of airborne allergens. 3 In this context, pollen is a typical outdoor aeroallergen that is an independent risk factor for the development of allergic respiratory diseases; 4 with about 30-58% of AR patients have been shown to be sensitized to pollen. Studies investigating the effect of nitrogen dioxide (NO 2 ) and sulphur dioxide (SO 2 ), two major fossil fuel-derived air pollutants, have demonstrated that these increase the allergenicity of pollen, 5 thus possibly aggravating or inducing AR in susceptible individuals.. Black carbon (BC) produced by the incomplete combustion of fuel is an important constituent of atmospheric particulate matter (PM) and is the second-largest contributor to global warming. 6 When emitted into the atmosphere, BC undergoes an aging process during which its particle morphology, chemical features, and redox activity may change. 7 In the present study we have investigated the alterations in the transcriptome in human nasal epithelial cell (hNECs) exposed to BC and pollen allergen in vitro.

Isolation and cultivation of human nasal epithelial cells (hNECs)
Nasal epithelial tissues were obtained from seven patients undergoing nasal surgery (turbinoplasty or septoplasty). Nasal polyps and the nasal tissues from patients with allergies or other chronic epithelial diseases were excluded. None of the patients had other systemic diseases or had received glucocorticoids or antibiotics within 3 months before the study. The study protocol was approved by the Ethics Committee of Beijing Tongren Hospital, and all patients provided written informed consent prior to any samples being taken for investigation.
Freshly obtained nasal mucosal samples were washed in phosphate buffer saline (PBS) with 200 U/mL penicillin and 50 mg/mL streptomycin, and then incubated overnight at 4 °C in 0.1% pronase (Protease XIV; Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco's modi ed eagle media (DMEM) culture medium to enzymatically digest the tissue. Following incubation, the separated epithelial cells were collected and washed by centrifugation at 100 g for 5 minutes and re-suspension in fresh DMEM. The washed cells were seeded at a concentration of 1 × 10 6 cells on porous membrane inserts (Corning® Transwell polycarbonate membrane inserts, 0.4 µm; 6.5 mm diameter; Corning Inc., N.Y., USA) coated with 150 µl collagen I (66 ng/ml; Sigma-Aldrich, St. Louis, Mo., USA), and cultured at 37 °C in 5% CO2 in air atmosphere. Once the cultures had reached 70-80% con uence by day 4, the culture medium was removed from the inserts, and BEGM: DMEM (1:1) medium was added to the basolateral side (insert wells) to differentiate cells. The cell cultures were assessed for transepithelial resistance (TER) using Millicell-ERS Volt-Ohm Meter (Millipore, Temecula, CA, USA). When the TER of individual cultures exceeded 2000Ω × cm 2 , the cultures were established as air-liquid interface (ALI) cultures, and subsequently used to assess the effects of exposure to BC and pollen.

BC and pollen preparation and exposure
Ozone-oxidized black carbon (BC), which is consistent with black carbon in the real environment, 8 was obtained from State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University. Prior to use, the BC was dissolved in deionized water at a concentration of 2 mg/ml and sonicated for 10 min using an ultrasonicator.
Artemisia annua pollen were collected from Beijing and aqueous protein extracts of the pollen were prepared by resuspending 2 g of pollen grains in 35 mL PBS buffer (0.14 M NaCl, 2.7 mM KCl, 7.8 mM Na2HPO4, 1.5 mM KH2PO4) and shaking for 12 h at 4 ℃. The supernatants were collected for estimation of the protein concentration, using the bicinchoninic acid (BCA) method (Thermo Fisher Scienti c, Carlsbad, CA, USA). The pollen protein was then dissolved in deionized water at a concentration of 10 mg/ml.
The cells on Transwell membranes of ALI cultures were washed twice with sterile PBS, and the supernatant was removed. The membranes were exposed to either doubling concentrations ranging 12.5 to 300 µg/ml or doubling concentrations of pollen protein ranging from 50 to 300 µg/ml for 24 h, and following exposure, the medium from the basolateral compartment was transferred to a 1.5 ml vial and stored at − 20 °C until further analysis. The cells were trypsinized off from the membrane, lysed in RLT buffer (RNeasy mini kit, Qiagen, Ven-lo, The Netherlands), and stored at − 80 °C until further analysis.

Cell viability
Cytotoxicity effects of BC and pollen in hNECs were determined using the Cell Counting Kit-8 (CCK8 CK04-500T Dojindo, Japan), according to manufacturer's protocol. Cells (5 × 10 4 /well in 96-well plates) were exposed to BC and/or pollen for 24 h at 37 °C. 10 µL CCK8 solution was added to each well and the cells were incubated for a further 2 h at 37 ℃. At the end of this incubation, the uorescence of individual wells was determined at 450 nm using uorescence microplate reader (Hitachi, Ltd., Tokyo, Japan).

High-throughput RNA sequencing (RNA-Seq)
For the transcriptomic studies, ALI-hNECs were treated with 25 µg/ml BC ± 200 µg/ml pollen for 24 h at 37 °C in CO 2 incubator. At the end of incubation the hNECs were harvested and total RNA was extracted from the cells using RNeasy mini kit 147 ((Qiagen, Germany)). All RNA samples were stored at -80 °C until use. For each experiment, three different batches of cultured cells were used and assays were performed in triplicate.
Paired-end libraries were synthesized by using the TruSeqTM RNA Sample Preparation Kit (Illumina, USA) following TruSeqTM RNA Sample Preparation Guide. Brie y, the poly-A containing mRNA molecules were puri ed using poly-T oligo-attached magnetic beads.
Following puri cation, the mRNA was fragmented into small pieces by incubation with divalent cations at 94 °C for 8 min. The cleaved RNA fragments were copied into rst strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then underwent an end repair process; to add a single 'A' base and ligation of the adapters; before being puri ed and enriched with PCR to create the nal cDNA library. Puri ed libraries were quanti ed by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to con rm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10pM and were then sequenced on the Illumina NovaSeq 6000 (Illumina, USA).
The library construction and sequencing was performed at Shanghai Sinomics Corporation.
Quantitative Real-time reverse transcription(qRT-PCR) ALI-hNECs were exposed to 25 µg/ml BC ± 200 µg/ml pollen in the absence or presence inhibitors (100 µM NAC, 10 µM MCC950, or 50 µM YVAD) for 24 h at 37 °C in CO 2 incubator, and at the end of incubation total RNA was extracted from the cells using the TaKaRa

Statistical analysis
Data from at least three independent experiments were expressed as mean ± standard deviation (SD), and analyzed for signi cance using Spearman correlation coe cients, two-tailed indirect Student's t-test or one-way analysis of variance (ANOVA), followed by the LSD post hoc test for multiple comparisons. All statistical analyses were performed using SPSS 25.0 statistical software and a P value < 0.05 was considered statistically signi cant.
The parameter Q value was used for statistical screening of differential genes. The difference screening criterion was q value < 0.05, and FC2, that is, the change of the expression value was up 2 times (FC ≥ 2) or down 2 times (FC ≤ 0.5). Enrichment analysis of different genes based on Gene Ontology (GO) and KEGG database for each Gene, was additionally performed to determine the major biological functions and pathways of the different genes. 9,10

Transcriptome alteration and functional enrichment analysis
A subset of 58,300 human genes was assayed in this study. Gene expression changes were analyzed by comparison between BC, pollen and BC + pollen treated groups and the control groups using statistical criteria of > 2.0-fold changes with a p-value of < 0.05. Expression of 40 genes was signi cantly altered (17 genes up-regulated and 23 genes down-regulated) following exposure to 200 µg/ml pollen ( Fig. 2A), 21 genes signi cant altered (6 genes up-regulated and 15 genes down-regulated) following exposure to 25 µg/ml BC (Fig. 2B), and 114 genes signi cantly altered (34 genes up-regulated and 80 genes downregulated) following exposure to 200 µg/ml BC (Fig. 2C). Moreover, exposure to a combination of 25 µg/ml BC + 200 µg/ml pollen resulted in signi cant alterations in even greater number of genes (233 genes up-regulated and 60 down-regulated) (Fig. 2D).
Gene ontology (GO) biological processes and KEGG pathway enrichment analysis of the altered genes further demonstrated that no GO terms were substantially enriched in hNECs exposed to 25 µg/ml BC or 200 µg/ml pollen alone ( Fig. 3A and B). However, for hNECs exposed to 200 µg/ml BC, the up-regulated GO terms were mainly associated with hypoxia stress response (i.e., cellular response to oxygen species and response to hydrogen peroxide) (Fig. 3C); whereas the top enriched GO terms for hNECs exposed to a combination of BC (25 µg/ml) and pollen (200 µg/ml) were mostly associated with the in ammatory response (i.e., regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, degranulation and mast cells medical immunity, and phagocytosis) (Fig. 3D).

Gene expressions validation by RT-qPCR analysis
In order to con rm the results of microarray, RT-qPCR was applied to further examine the expressions of target genes. Pro-in ammatory factor IL1B was up-regulated in hNECs exposed to 25, and 200 µg/ml BC and 25 µg/ml BC + 200 µg/ml pollen (Fig. 4A). For in ammation related gene expressions, the primary regulator of mononuclear phagocytes, CSF-1 was over-expressed in hNECs exposed to 25 µg/ml BC and 25 µg/ml + 200 µg/ml pollen; whereas for mast cell mediated immunity related gene expressions, FCER1G was over-expressed in hNECs exposed to 200 µg/ml pollen and 25 µg/ml BC + 200 µg/ml pollen. Although these results were consistent with the microarray data, there was a small difference in terms of the degree of over-expression between microarray method and RT-qPCR assay, which may due to differential sample preparation and sensitivity of the measuring method.

Discussion
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 signi cantly 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 in ammatory 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 in ammatory process.
IL1B is a potent proin ammatory 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 in ux and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and broblast proliferation and collagen production. IL1R1(interleukin-1 receptor 1) as a member of IL-1 family cluster may regulate the process of in ammation 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 ndings from the present study.. Similarly, our ndings for differential expression of other genes in uencing neutrophils are consistent with other studies. For example, TGAM (integrin alpha M chain), a leukocyte-speci c 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-in ammatory molecules and increased oxidative metabolism by neutrophils 15 , which is consistent with our nding of the anti-oxidative and anti-in ammatory effect of S100A9 on neutrophils. An in vitro study has con rmed 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 in ammatory response induced by BC and pollen in hNECs.
Macrophages are indispensable as members of the innate immune system, as they regulate normal physiology as rst 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 in ammation. 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 nding 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 nding 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 ndings 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-in ammatory 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  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 in ammatory reactions associated with allergic disorders. This involves a chain of reactions following antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-a nity 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.

Conclusion
In summary, exposure to 25 g /ml BC and 200 g /ml pollen signi cantly decreases the viability of epithelial cells, and induces innate immune and allergic in ammation in hNECs. IL1B, CSF1, and FCER1 are main the upstream regulators in the in ammatory response induced by BC and pollen. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.