Analysis of Allergen Components and Identification of Bioactivity of HSP70 in Pollen of Populus Deltoides

doi:10.21203/rs.3.rs-541522/v1

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Analysis of Allergen Components and Identification of Bioactivity of HSP70 in Pollen of Populus Deltoides

https://doi.org/10.21203/rs.3.rs-541522/v1

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Background: Allergies caused by pollen from Populus deltoides are common, but the allergic components are still unclear.

Methods: The total proteins in pollen of P. deltoides were analyzed by proteomics, and the potential allergens were identified via the WHO/IUIS database and the allergenOnline database retrieval. The target protein was screened by bioinformatics and expressed in Escherichia coli. The biological activity of the expressed product was verified by animal experiments.

Results: 3929 total proteins in pollen of P. deltoides were identified, and 49 potential allergens belonging to 10 protein families were recognized by database retrieval. B9N9W6 protein of Hsp 70 family was screened by bioinformatics analysis and expressed successfully. ELISA showed that B9N9W6 can stimulate the immune system to produce specific IgE and promote the generation of IL-4. Flow cytometry showed that B9N9W6 can significantly stimulate the proliferation of CD4⁺T cells and promote the polarization of Th2 cells. The pathological sections of mice lung tissues indicated that alveolar destruction was more severe in the B9N9W6 group than that of extract group, and there were more inflammatory cells infiltration, mucus exudation and bleeding.

Conclusion: B9N9W6 is an important antigenic substance in the pollen of P. deltoides. Due to the conserved structure of Hsp 70 family, more attention should be paid to the possibility of sensitization when HSP 70 from any pathogenic species is administered.

Molecular Biology

General Biochemistry

Populus deltoides pollen

Proteosome

Hsp 70

Allergen

Allergic disease

Allergic asthma is a chronic airway inflammation disease, which characterized by chest tightness, shortness of breath, and coughing after exposure to allergens [1]. The incidence of allergic asthma has been increasing in recent years [2]. At least 300 million people suffer from allergic asthma worldwide [3], which is highest among children [4, 5]. The main pathogenesis of allergic asthma is the production of specific IgE antibody, chronic airway inflammation and airway hyperresponsiveness during the immune system response to allergens in the environment, accompanied by the imbalance of Th1/Th2 cells and other comprehensive factors [6–8].

Pollen from plants is an important source of air-borne allergens, which seriously affects the quality of life for people who is susceptible to allergies [9]. During the period of flower opening, pollen grains are released into the air to form biological aerosols; thus, individuals are inevitably exposed to pollen. Populus deltoides is widely cultivated in China due to its urban greening, windbreak, and sand-fixing berm. In May of each year, mature pollen of P. deltoides is densely suspended in the air, which causes a mass of pollen to frequently contact people’s eyes, nostrils, mouth and skin, leading to tears, sneezing, itching and other symptoms. Our recent studies demonstrated that mature pollen of P. deltoides extract contains antigenic substances with strong sensitization. However, allergic components in P. deltoides pollen remain largely unclear.

The identification and purification of pollen allergens is of great significance for pollen allergy disease, especially in diagnosis and allergen immunotherapy (AIT). In recent years, proteomic techniques have become powerful tools for comprehensive allergen analysis, such as Par h 1 in Parthenium hysterophorus, and Art an 7 in Artemisia vulgaris were identified by this method [10, 11]. In China, proteomics was used to analyze and compare the possible allergens in mutants of P. deltoides [12, 13]. Wang et al. have analyzed the possible allergen components in Populus Tomentosa by proteomics [14]. However, a systematic experimental basis is lacking for the identification of allergens in P. deltoides pollen.

HSP (Heat Shock Protein) 70 domain protein has a subtle relationship with allergic diseases. Previous studies have found that Hsp70 is an important mediator to mediate allergic reactions and is capable of binding IgE antibodies in allergic patients [15]. The levels of Hsp70 are significantly elevated in patients with allergic rhinitis [16]. Interestingly, Hsp 70 is widely present in plant pollen as a pan-allergen, which could be responsible for a part of the allergenic cross-reactivity between proteins from different pollens and plant food [17]. However, the biological function of Hsp 70 remains largely unknown.

To screen and verify P. deltoides pollen allergens, we analyzed the total protein of pollen through proteomics. Then, the sequences of identified protein were compared with the confirmed allergen via relevant allergen database to identify the allergen components in pollen. After that, Hsp 70 was expressed in prokaryotic expression system and explored its biological function by animal models. As far as we know, this is the first report of comprehensive allergenic proteins and Hsp 70 biological function in pollen of P. deltoides.

P. deltoides pollen sample

The pollen samples of P. deltoides used in this study were collected at the Yangtze River embankment (Wuhu, China) from Apr 20 to May 20, 2018, and stored in a refrigerator at -80°C.

Experimental animal

A total of 30 SPF female BALB/c mice (6-weeks) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (License number: 20170005030182). The animals were kept under Specific Pathogen Free (SPF) laboratory conditions in the Hangzhou Hibio Technology Co. Ltd, with the room temperature at 22–26°C, light and darkness for 12 hours, respectively. Animals were free to eat and drink.

Protein extraction

Protein preparation for proteomics was as follows: The pollen samples were treated with trichloroacetic acid with a final concentration of 20% at 4°C for 2 h, centrifuged at 12000 g at 4°C for 3 min, and the supernatant was discarded. The protein precipitate was washed three times with pre-cooled acetone and reconstituted with 8 M urea. Then, the proteins were analyzed by 12% SDS-PAGE. Protein preparation for animal experiments was as follows: Dried pollen was extracted with PBS (solid-liquid ratio of 1 g: 20 mL) at room temperature (RT) for 24 hours. The extracted materials were precipitated at RT for 8 hours. The crude extracts were filtered through 20 µm filter paper. Then, the extracts were sterilized through a 0.22 µm strainer (Millex GP) and stored at 4°C. Finally, the protein concentration was measured by the BCA kit (Sangon, C503071-0250).

Proteomics analysis of P. deltoides pollen

Proteomics analysis was performed with reference to relevant study methods [18]. The main process was as follows: total protein was digested with trypsin, and the tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column. The tryptic peptides were analyzed via LC-MS/MS, and. We map and annotate the obtained MS/MS data using Maxquant software (v1.5.2.8). Subsequently, all the annotated peptide data were classified by R software for gene ontology classification and KEGG signaling pathway enrichment. The thresholds for the identification of total proteins and potential allergens in P. deltoides pollen were determined by relevant literature [19].

Screening of potential allergens in the Hsp70 protein family

The potential allergens of the Hsp70 protein family were screened by following steps. First, we performed T/B cell epitopes prediction for potential allergens of the Hsp70 protein family using the Immune Epitope Database and Analysis Resource (IEDB) ( http://www.iedb.org/ ). Then, Antigenicity of the HSP70 were predicted through Vaxijen v2.0 (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) (Threshold 0.4). The formula, molecular weight (MW), stability and isoelectric point (PI) for B9N9W6 were predicted via ExPASy (http://web.expasy.org/protparam/), and its solubility was analyzed by ProtScale (http://web.Expasy.org/protscale/). Thirdly, we performed sequence alignment with HSP70s through the known allergens reported in the WHO/IUIS database and AllergenOnline database, respectively. Finally, we used Swiss-model for homology modeling to screen out Hsp70 with the highest surface complementarity with IgE antibody.

Prokaryotic expression and purification of Hsp70

Availability of adequate amounts of the pure allergens is essential to further understand their molecular structures, which is a pre-requisite for the development of more efficacious allergen immunotherapy. The amino acid sequences and coding genes of Hsp 70 were retrieved from Uniprot database. The gene of Hsp 70 was synthesized and cloned into prokaryotic expression vector pET-28a (+). The expression of His-tagged recombinant Hsp 70 in transformant E. coli BL21 was induced by IPTG (final concentration = 1 mmol/L), and the expression products were analyzed by SDS-PAGE. Then, the bacteria were collected and disrupted by sonication. Hsp 70 was purified by Ni-NTA protein purification kit (Sangon, C600320-0001), and protein concentration was measured by the protein concentration assay kit (Sangon, C503071-0250).

Preparation of asthmatic model

All BALB/c mice were randomly divided into 3 groups (10 mice in each group): PBS group, pollen extract group, and Hsp 70 group. Mice in extract group and Hsp 70 group were injected intraperitoneally with 10 µg pollen extract or Hsp 70 [dissolved in 4% (W/V) Al(OH)₃ in PBS, endotoxin-free] on 0, 7, and 14 days, respectively, to sensitize. From the 21st day, 0.5 µg/ml pollen extract or Hsp 70 suspension was sprayed for excitation for 30 min/time for one week. The PBS group was replaced with PBS containing 4% (W/V) Al(OH)₃. Mice in each group were injected intraperitoneally with 0.8 mg BrdU (dissolved in PBS) before the last challenge. After 24 hours, blood was taken from the eye socket, left at 4°C for 2 hours, and centrifuged at 4000 g for 5 minutes. The collected serum was stored at -80°C.

Enzyme-linked immunosorbent assay (ELISA)

The levels of total IgE antibody and IL-4 in serum were measured by ELISA kits from Songon, Inc. and Jianglaibio (Shanghai, China) (Sangon, D721094 \ Jianglaibio, JL20266) according to the manufacturer’s protocol.

Flow cytometry

The spleens of mice in each experimental group were aseptically collected, and splenic cell suspension was prepared. The proliferation of CD4⁺ T cells and the ratio of Th1/Th2 in spleen cell suspension were detected according to the reported method [20]. In brief, the Th1/Th2 ratio in the splenic cell suspension was detected by the Th1/Th2 flow cytometry kit (Lianke Biotech, No. 70-KTH201) according to the kit’s instructions. Cell suspension was incubated for one hour at RT in the dark after adding Anti-mouse CD4-FITC antibody (Invitrogen, No. 11-0042-85) and Brdu-PE antibody (Invitrogen, No. 12-5071-41), respectively, and then determined by flow cytometry.

Histological analysis

After the mice lung tissues were embedded in paraffin, sectioned (5µm thick), and hydrated with a series of alcohol, stained with hematoxylin and eosin (H&E), and then the changes in lung tissue structure were observed with a Nikon Ni-E microscope. Images were captured with a Nikon DS-Ri2 camera.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD). Data analysis was performed with SPSS16.0 statistical software package. One-way analysis of variance (ANOVA) was used for the comparison of multiple sample means, and the least significant difference (LSD) method and Tamhane’s T2 method were used for the comparison between the two samples. The statistical test level α = 0.05.

Definition of P. deltoides pollen proteome

In the present study, we have used workflow (Fig. 1) to classify its allergen families and to characterize new P. deltoides pollen allergen candidates. Mass spectrometry data of the P. deltoides pollen proteome showed that the protein mass was mainly concentrated within 150kD, and the protein sequence coverage was mainly below 40% (Fig. 2A). The quantitative analysis of peptide length showed that peptides were mainly distributed in the length of 7–27 amino acid residues, and the number of peptides with length 7-17AA was relatively concentrated (Fig. 2B).

Analysis by LCMS/MS showed that a total of 3, 929 different proteins were identified. Gene ontology revealed that in terms of biological processes, these peptides were mainly involved in the metabolic process (32.49%), cellular process (27.26%) and single-organism process (19.21%) (Fig. 3A). From the perspective of cell components, it mainly involves cell (38.83%), organelle (23.74%), membrane (16.63%) and macromolecular complex (16.09%) (Fig. 3B). As for molecular functions, 43.35% of peptides were involved in catalytic activity, and 43.32% of peptides were related to binding (Fig. 3C). The subcellular localization results showed that 37.47% of the peptides were located in the chloroplast, 28.86% of the peptides were located in the cytoplasm, and 14.07% of the peptides were located in the nucleus (Fig. 3D).

Enrichment analysis of the KEGG signaling pathway and domains contained peptides

Enrichment analysis on the 3929 identified peptides showed that the top 5 signal pathways that were significantly enriched were ribosome, carbon metabolism, biosynthesis of amino acids, citrate cycle, and proteasome (Fig. 4A). The domain enrichment results showed that the top three domains were Aldolase-type TIM barrel, Heat shock protein 70kD C-terminal domain and Translation protein beta-barrel domain (Fig. 4B).

Allergenic protein families and candidate allergenic proteins

Sequence alignment between the WHO/IUIS database and the allergenOnline database identified 49 potential allergens that belonged to 10 protein families (Table 1).

Table 1

The information of predicted allergenic proteins in the pollen of *P. deltoides*
Allergenic protein family	Protein accession	Identity (%)	Protein description	Sum
pathogenesis-related proteins	A9PHF7	Hev B 6 (95.2%)	Class 4 pathogenesis-related family protein	1
Profilins	A9P8N4	Hev b 8 (94.7%)	Profilin	1
Peroxidases	B9GYJ9	Che a 3 (61.8%)	Peroxidase	1
Seed storage proteins	A0A2K1ZNE2 B9H8M2 B9HNV4	Gly m Bd (62.2%) Pis v 2 (78.9%) Ana o 2 (75.3%)	Vicilin-like seed storage protein 11S globulin seed storage protein Vicilin-like seed storage protein	3
Proteases	A0A2K1XV49 A0A2K1ZSB7 A0A2K2AML0 A9PA38 A9PBM7 A9PCK2 B9GJE2 B9GR19 B9GWZ3 B9H185 B9H290 B9HR73 B9I8Y7 B9IKK0 B9MUK8 U5GXJ8	Der p 15 (57.3%) Pen c 1 (57.6%) Ana c 2 (77.0%) Der p 15 (56.6%) CPA63 (69.4%) CPA63 (68.5%) Per a 2 (61.1%) CPA63 (63.5%) CPA63 (68.6%) Pen n 13 (51.8%) Cup s 1 (57.5%) Pen n 13 (51.7%) CPA63 (62.9%) Pen n 13 (56.9%) Asp f 1 (59.6%) Pen n 18 (57.2%)	Protease Do-like 2 Subtilisin-like protease Cysteine protease ATP-dependent Clp protease Aspartyl protease Protein Protein aspartic protease in guard cell Aspartic protease family protein Aspartyl protease family protein Aspartyl protease family protein Subtilisin-like protease Kunitz-type protease Subtilisin-like protease Aspartyl protease 26S protease regulatory subunit 7 family protein 26S protease regulatory subunit 7 family protein Subtilisin-like protease	16
Expansins	A0A2K1Z7R5 A9PEQ7 B9H521 U5FK15 U5GIP5	Pas n 1(68.1%) Sor h 1(55.0%) Lol p I (57.9%) Cyn d 1(60.8%) Dac g 1(65.2%)	Expansin-like A2 Expansin family protein Alpha-expansin 11 family protein Expansin S1 family protein Expansin-like B1	5
Calcium-binding proteins	A9P988 A9PCU6	Jun o 4 (69.6%) pollen allergen group 2	Probable calcium-binding protein Calcium-binding EF hand family protein	2
Inhibitory proteins	A9P9T5 B9GNE2 B9GX99 B9H290 B9HSW8 B9I4L9	Ani s 4 (64.0%) Api m 6 (79.5%) Ani s 4 (58.8%) Cry j 1(56.1%) Pla o 1 (63.3%) Ole e 3 (75.9%)	Cysteine proteinase inhibitor Cysteine proteinase inhibitor Cysteine proteinase inhibitor Kunitz-type protease inhibitor Cell wall /vacuolar inhibitor of fructosidase Invertase/pectin methylesterase inhibitor family protein	6
Polygalacturonase	B9GQC0 B9H9W1	Zea m 13 (55.4%) Cry j 2 (71.4%)	Polygalacturonase-like family protein Polygalacturonase	2
Heat shock protein 70	B9GEL5 B9GJ14 A0A2K1XBF6 B9HMG2 B9HMG7 B9HMG8 B9HN74 B9HTJ7 B9HV59 B9N9W5 B9N9W6 B9NBF4	Alt a 3 (62.6%) Der p 28 (77.4%) Cor a 1 (49.6%) Pen c 19 (90.1%) Pen c 19 (88.7%) Pen c 19 (91.6%) Pen c 19 (78.1%) Pen c 19 (90.6%) Pen c 19 (70.9%) Der p 28 (88.2%) Pen c 19 (90.0%) Pen c 19 (91.8%)	Heat shock protein 70 Heat shock protein 70 Activator of 90 kDa heat shock protein ATPase homolog Heat shock protein 70 Heat shock protein 70 cognate Heat shock protein 70 cognate Heat shock protein 70 Heat shock protein 70 Heat shock protein 70 Heat shock protein 70 cognate Heat shock protein 70 cognate Heat shock protein 70 cognate	12
Note: Only homologous allergen with the highest identity has been displayed.
Identity, homologous allergen in the AllergenOnline database. Sum, total potential pollen allergens identified in the family.

Screening of potential allergens in the Hsp70 protein family

All the Hsp70s were checked, and B9N9W6 was selected as a potential allergen to elicit host immune response due to its physical and chemical characteristics as follows. IEDB epitope prediction showed that had 3 T-cell epitopes and 4 B-cell epitopes with high affinity (Table 2, 3). The formula of B9N9W6 was C₃₁₄₆H₅₀₆₂N₈₈₀O₁₀₀₁S₂₁, MW was 71 900, PI was 5.34, and antigenicity was 0.5714 (Threshold > 0.4), which was considered as a stable hydrophilic protein. Query through AllergenOline database showed that B9N9W6 had a conserved Hsp70 domain. Protein homology analysis revealed that the Hsp70 family protein sequences were extremely conserved (Fig. 5). Secondary structure of B9N9W6 was dominated by α-helix and β ladder (Fig. 6), and it might be a hetero-dimer according to the crystal structure on the surface (Fig. 7).

Table 2

Prediction of T cell epitopes in B9N9W6
Epitope peptide	IC₅₀ (SMM)	IC₅₀ (NN)
QDLLLLDVTPLSVGI	25	21.2
LNVLRIINEPTAAAI	99	19.1
LRIINEPTAAAIAYG	176	34.5
Note: The predicted output is given in units of IC₅₀nM for Stabilized Matrix Method (SMM) and Neutral-Network (NN) scoring. The lower number indicates higher affinity. Peptides with IC₅₀ values < 50 nM are considered high affinity.

Table 3

Prediction of B cell epitopes in B9N9W6
Epitope peptide	Position
GRRFSDPSVQSDMKHWPF	78–95
KASGVKNKITITNDKGRLGKDDIERMVQEAERYKAEDEKVKKKVEAKNA	499–547
NTVRDDKVGGKLDPADKQKIEKEIEETIDWLDRNQLAEVDEFEDK	557–601
QGAGGDVPMGGGAQMPGGAYSKASSGGSGAGPKI	618–651
Note: Bepipred Linear Epitope Prediction 2.0 was selected to predict the B cell Epitope, which analyzed the sequence characteristics of antigen and used the size of amino acid residues to predict the B cell epitopes.

Protein expression and purification of B9N9W6

SDS-PAGE of B9N9W6 showed specific band with a MW at 70KD, and purified product showed a clear band at same position (Fig. 8). His-tag detection revealed that the protein purity was more than 90%.

B9N9W6 induced antibody and cytokine responses

In order to explore the effects of B9N9W6 on serum antibodies and cytokines, the levels of IgE and cytokine IL-4 in serum were detected by ELISA kit. The results were showed in Fig. 9. The IgE level in the extract group (13.83 ± 5.59 ng/mL) was significantly higher than that in the PBS group (7.58 ± 2.40 ng/mL, P < 0.05), but significantly lower than that in B9N9W6 group (25.76 ± 11.90) ng/mL, P < 0.01). In terms of IL-4 level, the IL-4 level in the extract group (120.08 ± 36.58 pg/mL) was higher than that in the PBS group (74.69 ± 25.30 pg/mL, P < 0.05), while the IL-4 level in the B9N9W6 group (255.24 ± 81.88 pg/mL) was the highest among all groups (P < 0.01).

B9N9W6 induced CD4⁺ T cell proliferation and Th2 differentiation

In order to evaluate the ability of B9N9W6 to stimulate the proliferation of CD4⁺ T cells, we detected the number of proliferating CD4⁺ T cells and their subsets by flow cytometry. The results showed that compared with the PBS group (12.84 ± 0.53 %) and extract group (18.10 ± 0.58 %), B9N9W6 significantly promoted the proliferation of CD4⁺ T cells (27.86 ± 1.07 %, P < 0.01) (Fig. 10A, C1). Compared with the PBS group (2.39 ± 0.34), the ratio of Th1/Th2 in extract group (1.75 ± 0.24) and B9N9W6 group (1.69 ± 0.21) had significantly lower (P < 0.01) (Fig. 10B, C2).

B9N9W6 induced allergic inflammation in the lung tissue of mice models

Compared with the PBS group, the extract group showed reduced lung tissue alveoli due to destroyed anatomical structure, and increased mucus in the respiratory bronchus (Fig. 11B). A large number of inflammatory cells were exudated to the lung field. Alveolar septum thickens due to edema while mucus and inflammatory cells appeared in the terminal bronchioles and its periphery (Fig. 11E, H). Compared with the extract group, a mass of red blood cells exudate was found in the B9N9W6 group, and the destruction of alveoli was further aggravated (Fig. 11C); Bullae formed, the collapsed alveolar cavity and exudate of erythrocyte were detected; Mucus and inflammatory cells were abundant in the terminal bronchioles (Fig. 11F, I).

In recent years, allergic diseases caused by pollens have attracted much attention, especially in the plant species releasing pollens, specific IgE reactivity and the influence of air pollutants on pollen transmission [21–23]. Nevertheless, identification of allergenic proteins and their bioactivity have remained elusive. In this study, we detected 3929 distinct proteins in pollen of P. deltoides by proteomics. We performed systematic bioinformatics analysis of all identified proteins, including protein annotation, functional classification and functional enrichment. Through functional annotation, we found that the total proteins of P. deltoides pollen and P. tomentosa pollen have great difference in function [14]. By GO categorization, we found that the total proteins of P. deltoides pollen were significantly different from those of its two mutants in biological process, molecular function, and cellular component [12]. KEGG enrichment analysis indicated that these identified proteins were not only involved in organelle composition and biogenesis, but also in biological processes such as metabolism and synthesis. There are great differences in the ingredients and functions of pollen proteins among different species of the same genus, which provides a material basis for antigen screening.

Plant-derived allergens mainly belong to disease-related protein 10 (DRP-10), thomas protein-like protein (TLP), Non-specific lipid transfer proteins (nsLTPs), expansion proteins, calcium binding proteins and profilin protein families [24, 25]. These proteins are called pan-allergen in specialized terms such as profilin, because the same family of proteins has a common antigenic determinant, and they can cause a wide range of cross-reactions [26]. Proteins of the same family share a common domain and are relatively conservative in structure, which caused the common allergen proteins can be identified in a variety of plants [27–29]. In this study, through sequence alignment in the database, we identified 49 potential allergens belonging to 10 protein families. The protease family and the Hsp70 family are the most abundant. These results indicated that P. deltoides pollen not only contained abundant protein components, but also easily had fruit-vegetable-pollen cross-reactive allergy syndromes.

Hsp represent a family of molecular chaperones that response to refolding proteins, protein trafficking, and cell signaling processes [30–32]. Hsp 70 is an important member of the Hsp family involved in stress response, which often used as a potential biomarker, therapeutic target, or modulator of inflammation [33, 34]. Furthermore, Hsp 70 is also involved leaf remodeling, flowering and disease resistance in plant [35–37]. A correlation between biological function and allergenic capacity of proteins related to stress response has not been clearly demonstrated. Studies had shown that luminal binding protein of Hsp 70 family in hazel pollen is a cross-reactive allergen [17]. C-terminal region of Hsp 70 of Echinococcus Granulosus is antigenic molecule inducing both B and T cell responses [15]. Coincidentally, a large number of proteins and potential allergens containing the Hsp70 domain were identified in this study, such as B9N9W6. Epitope prediction suggested that B9N9W6 might have antigenic activity. Sequence alignment showed that B9N9W6 was highly consistent with the amino acid sequences of known allergens Cla h 4, Der f 28, etc. Homology modeling for B9N9W6 found that its 3-dimensional structure was also highly similar which consists of two main useful realms separated by a hinge region (Fig. 6A, and 6B); which accorded with the structural characteristics of the Hsp70 family [38]. This remarkable conservation of both surface residues and main chain conformations in the Hsp 70 family plays an important role in conservation of IgE-binding epitopes[39].

Identification and purification of pollen allergens is of great significance both for the study of cross-allergic reaction and AIT. The immunoreactivity of Hsp 70 had been demonstrated in previous studies [17, 39, 40]. In this study, the bioactivity of B9N9W6 was detected by animal model. First, we demonstrated that B9N9W6 can stimulate the immune system to produce high levels of IgE antibodies and promote the production of IL-4 via ELISA. Allergen specific IgE antibody is a major cause of type I allergic diseases, such as asthma [41], IL-4 is an important proinflammatory factor secreted by Th2 cells to mediate allergic airway inflammation [42]. Meanwhile, the significant increase of IL-4 concentration in the B9N9W6 group indicated the imbalance of Th1/Th2 cells and the increase of Th2 cells. Secondly, we detected the proliferation of B9N9W6 to stimulate CD4⁺ T cells and their subgroup Th1/Th2 cells by flow cytometry. It was found that B9N9W6 could significantly stimulate CD4⁺ T cell proliferation and promote Th2 cell polarization. These results suggested an immunogenicity of B9N9W6, which were consistent with ELISA. All above results suggest that B9N9W6 may induce allergic inflammation in the airway of the mice model. To verify our hypothesis, the presence of inflammation was observed through H&E staining sections of the mice lung tissues. More inflammatory cells infiltration and mucus exudation were observed in the lung tissue of B9N9W6 group; the alveolar rupture was the most serious, and even caused pulmonary hemorrhage. Therefore, we consider that under the same concentration, the sensitization of B9N9W6 was stronger than that of extract. There are three possible reasons for this result: 1) B9N9W6 might have a dominant T/B cell epitope, which can strongly stimulate the immune response (Table 2, 3). 2) As a member of the Hsp 70 family, B9N9W6 could enhance the activity of antibody-presenting cells (APCs) in the process of antigen processing and presentation, which is essential for the initiation and modulation of the asthmatic immune response [43]. 3) Hsp 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in allergic airway inflammation [44]; the pathological findings of this study supported this view. Therefore, our findings suggested that B9N9W6 potentially induces allergen induced Th2 inflammatory responses.

In summary, This study empolyed the proteomics method to screen out 49 allergens from 10 protein families of P. deltoides pollen. Furthermore, B9N9W6 protein of HSP70 family was confirmed to has strong allergen activity and can induce allergic asthma in animal models. Our conclusions not only enrich the theoretical study of Hsp70 family in pollen allergens, but also provide reference for the study of cross-allergic reaction and immunotherapy of allergic diseases. In the future, we will focus on several candidate allergens that occured in KEGG enrichment analysis, especially in Hsp70 mediated signaling pathways.

Hsp: Heat shock protein; AIT: Allergen immunotherapy; DRP-10: Disease-related protein 10; TLP: Thomas protein-like protein; nsLTPs: Non-specific lipid transfer proteins; SPF: Specific pathogen free; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Gnens and Genomes

Statement of Ethics

This study was conducted in strict accordance with the proposals of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the mouse in this study were reviewed and approvednimal by the Institutional A Care and Use Committee of Hangzhou Hibio Technology Co.Ltd.(IACUC protocol number: HBFM3.68-2015). The pollen of P. deltoides used in this study were conducted in strict accordance with the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Disclosure Statement

The authors declare that they have no conflict of interest.

Funding Sources

This study was supported by National Natural Science Foundation of China, Grant Number 31971562.

Author Contributions

Wei Guo and Yilong Xi conceived the idea and wrote the manuscript. Xiaodong Zhan depicted figures and analyzed data. Feng Jiang contributed for revision. Yilong Xi contributed for overall editing and supervision. All authors approved its submission.

Acknowledgement

We would like to thank Hangzhou Hibio Technology Co. Ltd. for providing us with the SPF animal room and flow cytometer for this study.

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You are reading this latest preprint version

Analysis of Allergen Components and Identification of Bioactivity of HSP70 in Pollen of Populus Deltoides

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Experimental animal

Protein extraction

Screening of potential allergens in the Hsp70 protein family

Prokaryotic expression and purification of Hsp70

Preparation of asthmatic model

Enzyme-linked immunosorbent assay (ELISA)

Flow cytometry

Histological analysis

Statistical Analysis

Results

Enrichment analysis of the KEGG signaling pathway and domains contained peptides

Allergenic protein families and candidate allergenic proteins

Screening of potential allergens in the Hsp70 protein family

Protein expression and purification of B9N9W6

B9N9W6 induced antibody and cytokine responses

B9N9W6 induced CD4⁺ T cell proliferation and Th2 differentiation

B9N9W6 induced allergic inflammation in the lung tissue of mice models

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Analysis of Allergen Components and Identification of Bioactivity of HSP70 in Pollen of Populus Deltoides

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Experimental animal

Protein extraction

Screening of potential allergens in the Hsp70 protein family

Prokaryotic expression and purification of Hsp70

Preparation of asthmatic model

Enzyme-linked immunosorbent assay (ELISA)

Flow cytometry

Histological analysis

Statistical Analysis

Results

Enrichment analysis of the KEGG signaling pathway and domains contained peptides

Allergenic protein families and candidate allergenic proteins

Screening of potential allergens in the Hsp70 protein family

Protein expression and purification of B9N9W6

B9N9W6 induced antibody and cytokine responses

B9N9W6 induced CD4+ T cell proliferation and Th2 differentiation

B9N9W6 induced allergic inflammation in the lung tissue of mice models

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

B9N9W6 induced CD4⁺ T cell proliferation and Th2 differentiation