Upregulated Expression of Toll-Like Receptor 7 in Peripheral Blood Basophils of Patients With Allergic Rhinitis

Background Recently, it has been reported that Toll-like receptor 7 (TLR7) agonists can improve allergic rhinitis (AR) symptoms by up-regulation of Th1 cytokine release and suppression of Th2 cell functions. However, little is known of the expression of TLR7 in basophils of AR. Objective To explore the expression of TLR7 in basophils of AR, and influence of allergens on TLR7 expression. Methods The expression levels of TLR7 in basophils of patients with AR were determined by flow cytometry, and the influence of allergens on TLR7 expression was examined by real time (q) PCR. Results The percentages of TLR7+CCR3+ cells (P < 0.001 and P = 0.011), TLR7+CD123+HLA-DR− cells (P = 0 .016 and P = 0.042) and TLR7+CCR3+CD123+HLA-DR− cells (P = 0.046 and P = 0.035) in blood granulocyte and mononucleated cell populations of the patients with AR were increased, respectively compared with HC subjects. TLR7 MFI on CCR3+ cells (P = 0.050 and P = 0.043), CD123+HLA-DR− cells (P < 0.001 and P = 0.002) and CCR3+CD123+HLA-DR− cells (P < 0.001 and P = 0.003) were enhanced compared with HC subjects. Allergens Der p1 and OVA provoked upregulation of TLR7 expression at both protein and mRNA levels and IL-13 production in KU812 cells. House Dust Mite extract (HDME), Artemisia sieversiana wild allergen extract (ASWE), IL-31, IL-33, IL-37, and TSLP provoked elevation of IL-6 release from KU812 cells following 2 h incubation period. Conclusions The percentage of TLR7+ basophils and TLR7 expression intensity in a single basophil are both increased in the blood of patients with AR, indicating that basophils likely contribute to the pathogenesis of AR via TLR7.


Background
AR is consideredas an immunoglobulin E-mediated in ammation of the upper airway, which is a common condition with increasing prevalence.The pathogenesis of AR is multi-factorial and complex.There is an in ltration by in ammatory cells, particularly Th2 cells, eosinophils and basophils into nasal mucosal tissue that results in the allergic response.Over the last 20 years, the majority of studies on AR have been focused on proin ammatory cytokines.However, little is known about the expression of Toll-like receptor (TLR)7 in AR.
TLRs are vital elements of the mammalian immune system, bridging innate and adaptive immunity.TLR7, a member of TLR family, is an intracellular receptor expressed on the membrane of endosomes, which recognize single stranded RNA, signi cantly participate in the amelioration of AR symptoms.For instance, a clinical trial study on the Swedish population with AR revealed that AZD8848 as TLR7 agonist resulted in up-regulation of Th1 cytokines/chemokines (CXCL10, TNFα, IL-6, IFNγ) and suppression of Th2 cell functions in a pro le which results in an improvement of AR symptoms.Intranasal application of a TLR7 agonist GSK2245035 was reported to enhance expression of the Th1 cytokines and suppression of AR symptoms.The important roles played by TLR7 may be con rmed by genetic investigations which revealed the signi cant association between genetic variations in the TLR7 genes and AR.However, little is known of the expression of TLR7 in basophils in AR.
Basophils are known as primary effector cells of allergy, which are the least common granulocytes in peripheral blood of human beings.Apart from their contribution to allergy as initiators of IgE-induced acute reactions, basophils have shown elevated expression of thymic stromal lymphopoietin (TSLP) receptor in patients with AR after allergen stimulation, indicating that basophils are likely to be involved in AR.In the present study, 3 combinations of cell membrane molecules are employed to represent basophils, namely CD123 + HLA-DR -cells, CCR3 + cells, and CCR3 + CD123 + HLA-DR -cells.
Allergens are the causative factors of allergy.Skin prick tests showed that house dust mite (HDM) is the main allergen for AR in Korea, whereas pollens were more prevalent in Europe.It was reported that Der f and Der p were the most prevalent aeroallergens in China.Since HDM major allergens can activate TLRs and upregulate proin ammatory cytokine expression, they may act on TLR7 on basophils.
The aim of the study is to investigate the expression of TLR7 in peripheral blood basophils of patients with AR, and the in uence of airborne allergens on TLR7 expression in basophils.We observed that the percentages of TLR7 + basophils and TLR7 expression intensity in a single basophil are both increased in blood of the patients with AR.

Reagent
The following reagents were purchased from Biolegend (San Diego, USA): APC/Cy7-conjugated mouse anti-human CCR3, PE/Cy7-conjugated mouse anti-human CD123, PerCP/Cy5.5-conjugatedmouse antihuman HLA-DR.PE-conjugated mouse anti-human TLR7, PerCP-conjugated mouse anti-human TLR7 antibodies were obtained from Santa Cruz (Delaware Ave Santa Cruz, CA, USA).Human Fc receptor blocking solution, red blood cell lysis buffer, Zombie Aqua TM xable viability kit, and all isotype antibodies, Cyto x/Cytoperm™ Fixation/Permeabilization kits were obtained from BD Biosciences Pharmingen (Bedford, MA, USA).Fetal bovine serum (FBS, HyClone) and RPMI 1640 werepurchased from Gibco BRL (Grand Island, NY, USA).DSR6434 was purchased from R&D Systems (Minneapolis,MN).Ovalbumin (OVA, grade V), DNase Iand Trypan blue dye were purchased from Sigma-Aldrich (St Louis, MO, USA).Human IL-33, and human IL-37 were obtained from Peprotech (London, UK).Human IL-4, human IL-6, and IL-13 ELISA kits were bought form Cayman Chemical (Ann arbor MI, USA).Oligonucleotide primers for real time quantitative PCR (qPCR) were synthesized by Invitrogen Biotechnology Co. (Shanghai, China).Trizol reagent was obtained from Invitrogen (Carlsbad, CA, USA).Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from PANTM Seratech (Aidenbach, Germany).cDNAwas synthesized using iScript TM cDNA Synthesis kit.The resultant cDNA was subjected to qPCR that was performed with a LightCycler using a SuperScript III Platinum SYBR Green two-step qPCR kit.RNA was extracted by using a TaKaRa Mini BEST Universal RNA Extraction kit.cDNA was synthesized by using a PrimeScript RT reagent kit.Quanti cation of mRNA expression level was performed by applying TAKARA SYBR Premix EX Tag kit.Artemisia sieversiana wild allergen extract (ASWE) and Platanus pollen allergen extract (PPAE) were purchased from Macro Union Pharmaceutical Co. Ltd. (Beijing, China).Therecombinant full-length protein ofDermatophagoides pteronyssinus allergen 1 (Der p 1) expressed inE. coli wasobtained from Beijing Protein Innovation Co., Ltd (Beijing, China).House Dust Mite extract (HDME) I was supplied by ALKAbellÓ, Inc. (Denmark).Most of the general chemicals, such as salts and buffer components were of analytical grade.

Patients and samples
A total of 41 patients with AR and 22 healthy control (HC) subjects were recruited in the study.Their general characteristics were summarized in Table 1.The diagnosing criteria for AR in this experiment are in line with the 2015 AR Clinical Practice Guidelines issued by the American Academy of Otorhinolaryngology Head and Neck Surgery.Skin prick test was performed by using HDME I,ASWE and PPAE according to the manufacturer's instruction.Immediately after admission (acute exacerbation stage), the blood from each patient with AR was taken.Blood from HCs was collected in the outpatient clinic.From each individual, 10 ml of peripheral blood was taken into an EDTA-containing tube before centrifugation at 450 g for 10 min.The cells were used for ow cytometric analysis, and plasma was collected and frozen at −80℃ until use.

Flow cytometry analysis
To detect expression of TLR7 in human blood basophil cells, blood cells were challenged with or without ASWE,HDME or PPAE (all at a concentration of 1.0 μg/ml) for 60 min at 37℃, respectively, and 2 μg/ml brefeldin A was also added into the tube.Cells were then incubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie Green TM Fixable Viability kit for 15 min, and each labelled monoclonal antibody including APC/Cy7-conjugated mouse anti-human CCR3, PE/Cy7-conjugated mouse anti-human CD123, PerCP/Cy5.5-conjugatedmouse anti-human HLA-DR was added into the tube.
After red blood cells being lysed, resuspended leucocytes were xed and permeabilized using Cyto x/Cytoperm TM Fixation/Permeabilization kit according to the manufacturer's instructions.This was followed by adding a PE-conjugated anti-human TLR7 antibody into the tube and incubated at 4℃ for 30 min.
Finally, cells were resuspended in uorescence activated cell sorting (FACS)-ow solution and analysed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA).A total of 10,000 events in live cell gate were analysed for each sample.Data were analysed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA).Dead cells and doublets were excluded from analysis by live/dead cell dyes.

Enzyme-linked immunosorbent assay (ELISA)
Levels of IL-4, IL-6 and IL-13 in KU812 cell culture supernatant were determined by commercially available ELISA kits according to the manufacturer instruction.

RNA isolation and qPCR analysis
Total RNA was extracted from collected cells using a TaKaRa Mini BEST Universal RNA Extraction Kit. cDNA was synthesized by using a PrimeScript RT reagent Kit.The resultant cDNA was subjected to qPCR that was performed with a LightCycler System using a SYBR Premix Ex Taq Kit.The amplified product was detected by the presence of an SYBR green fluorescent signal.Each reaction contains 10 μl of 2x SYBR green Master Mix, 300 nM oligonucleotide primers, and 10 μl of the cDNA or plasmid DNA.-actin cDNA was used as the internal control and the ΔCt for all experimental samples were subtracted by the ΔCt for the control samples (ΔΔCt).The magnitude change of test gene mRNA was expressed as 2 −ΔΔCt .
The primers of TLR7 were forward: cct tga ggc caa caa cat ct and reverse: gta ggg acg gct gtg aca tt.

Statistical analysis
Statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA).
Human peripheral blood basophil data are displayed as a Scatter plot.Where Kruskal-Wallis analysis indicated signi cant differences between groups, for the pre-planned comparisons of interest, the paired Mann-Whitney U test was employed.KU812 cell data were expressed as mean ± SEM, and were evaluated by using a Student's t test.For all analyses, P < 0.05 was considered statistically signi cant.

Results
Expression of TLR7 in peripheral blood basophils of patients with AR It was reported that intranasal application of TLR7 agonist GSK2245035 suppresses AR symptoms.However, little is known about the expression level of TLR7 on basophils (one of the primary effector cells of allergy) in AR.We therefore examined expression levels of TLR7 in granulocyte populations and mononuclear cell populations of AR,respectively.The results showed that percentages of

Flow cytometry analysis of the expression of TLR7 in KU812 cells
In order to understand in uence of allergens on TLR7 expression in basophils, we investigated actions of Der p1, OVA, HDME and ASWE in KU812 cells.The results showed that only less than 3% of KU812 cells expressed TLR7 (Fig 3 ), and OVA at 1.0 μg/ml as well as Der p1 at 0.3 μg/ml induced upregulation of TLR7 expression in KU812 cells at 16 h following incubation (Fig 3C

qPCR analysis of TLR7 mRNA expression in KU812 cells
To con rm effect of allergens on TLR7 expression in KU812 cells, the expression of TLR7 mRNA in KU812 cellswas examined.It was found that OVA at 3.0 μg/ml and Der p1 at 1.0 μg/ml induced up to 2.1 and2.9-foldincreases in the expression of TLR7 mRNA over baseline control, respectively at 30 min following incubation (Fig4).DSR6434 failed to induce signi cant TLR7 mRNA expression in KU812 cells (Fig4).Apart from IL-37 (3 ng/ml) at 16 h following incubation, IL-31, IL-33 and TSLP all at 3 ng/ml, allergens HDME and ASWE both at 3 μg/ml did not induce signi cant increases in expression of TLR7 mRNA in KU812 cells following 30 min, 2 h and 16h incubation periods (Fig 4).
Levels of IL-4, IL-6 and IL-13 in the culture supernatantof KU812 cells In order to examine the actions of allergens, cytokines and TLR7 agonist in induction of cytokine release from KU812 cells, IL-4, IL-6 and IL-13 release from KU812 cells was investigated.The results showed that HDME and ASWE both at 3 μg/ml and plasma from HC provoked 2.13, 1.67 and Plasma from perennial AR allergic to dust mite alone (10 %) with or without HDME (3 μg/ml), plasma from seasonal AR allergic to artemisia alone (10%) with or without ASWE(3 μg/ml), plasma from seasonal AR allergic to Platanus pollen alone (10%) with or without PPAE(3 μg/ml) failed to alter IL-6 and IL-13 release from KU812 cells following 30 min, 2 h and 16 h incubation periods (data not shown).IL-4 is not detectable in the culture supernatant of KU812 cells (assay sensitivity: 2 pg/ml).

Discussion
It is found for the rst time that the percentages of TLR7 + cells are increased in blood CCR3 + , CD123 + HLA-DR -and CCR3 + CD123 + HLA-DR -granulocyteand mononucleated cell populations of the patients with AR, indicating that basophils likely contribute to the pathogenesis of AR via TLR7.However, CCR3 is not a speci c marker of basophils in granulocytepopulation, and a large proportion of eosinophils also express CCR3, suggesting that eosinophils may also be involved in AR through TLR7.
This may explain the observation that percentage of TLR7 + CCR3 + cells in blood granulocyte increased by 15.4 fold, whereas TLR7 + CD123 + HLA-DR -and TLR7 + CCR3 + CD123 + HLA-DR -cells enhanced only 6.9 and 4.9 fold, respectively in the patients with AR.In mononucleated cell population, apart from basophils, mononuclear phagocytes and CD34+ hematopoietic progenitor cells express CCR3, which may account for the nding that percentage of TLR7 + CCR3 + cells in blood mononuclear cells increased more than percentage of TLR7 + CD123 + HLA-DR -and TLR7 + CCR3 + CD123 + HLA-DR -cells in the patients with AR.
Similar degree of increases of TLR7 + CD123 + HLA-DR -and TLR7 + CCR3 + CD123 + HLA-DR -cells in mononucleated cell population of the patients with AR implicate that basophils in mononucleated cell population may also contribute to AR through TLR7.Moreover, the fact that TLR7 expression intensity of a single CCR3 + , CD123 + HLA-DR -and CCR3 + CD123 + HLA-DR -granulocyteor mononucleated cell increased in blood of the patients with AR also supports the view that basophils likely contribute to AR through TLR7.Since TLR7 is a therapeutic candidate target for allergic disorders,administration of TLR7 inhibitor AZD8848 can effectively improve the respiratory symptoms in patients with allergic asthma, our study provides important targeting cell basophil that TLR7 antagonists may act upon.Since basophils are well known primary effector cells in allergy, the current study may aid understand of the mechanism through which basophils are involved in the pathogenesis of AR.
It is not surprising that allergen extracts ASWE,HDME or PPAE have shown limited effects on TLR7 expression in basophils as these allergens normally uneasyto enter in blood and act directly on basophils.Thereport that HDM major allergens can activate TLRs on airway epithelium cellsand upregulate proin ammatory cytokineIL-6 and IL-8 expression may support our idea to look into effects of allergen extracts on TLR7 expression in basophils.Allergen stimulation did enhance percentage of TLR7 + CCR3 + cells in HC but did not show any signi cant effect on TLR7MFI on CCR3 + cells.This implicates that these allergens can increase proportion of TLR7expression CCR3 + cells in blood, but cannot enhance the density of TLR7expression on a single CCR3 + cell.
It is rather di cult to obtain large amount of highly puri ed inactive basophils, therefore a human basophil cell line, KU812 cells were employed to investigate the direct effect of allergens on basophils in the present study.Since KU812 cells have been shown to be a suitable model for studying the activation and degranulation of human basophils, we thought that these cells may be useful for study TLR7 expression and basophil activation.However, it was found that only up to 3% of KU812 cells expressing TLR7.Nevertheless, allergens Der p1 and OVA induced upregulation of TLR7 expression in KU812 cells at both protein and mRNA levels.The observation that TLR7 mRNA expression was enhanced by OVA at 2 h and Der p 1 at 30 min following incubation, and upregulated TLR7 protein expression provoked by them occurred at 16 h suggests that the transcription of OVA-and Der p1-induced TLR7 production is a relatively slow process in KU812 cells.As for TLR7 expression in primary blood basophils, KU812 cells showed little response to allergen HDME and ASWE challenge following up to 16 h incubation.These results may suggest that only certain types of allergens can alter TLR7 expression in basophils without relying on antigen-presenting cells.
Reduction of expression of TLR7 in KU812 cells by DSR6434 a potent agonist of TLR7 is an unexpected result.Nevertheless, a report that treatment of bone marrow-derived plasmacytoid dendritic cells with DSR6434 led to downregulation of TLR7 expression does suggest that DSR6434 can eliminate TLR7 expression on cells.Unexpectedly, IL-33 and IL-37 induced signi cant upregulation of TLR7 protein expression, but not signi cant TLR7 mRNA expression.It could result from an e cient transcription process of TLR7 protein in KU812 cells provoked by these two cytokines.Regulation of TLR7 expression by cytokines has been observed previously, which may support our current observation.
InductionofIL-6,but not IL-13 release from KU812 cells byHDME and ASWE, provocation of IL-13,but not IL-6release from KU812 cells by OVA and Der p1 are interesting results.Since IL-6 release and IL-13 secretion by KU812 cells are very likely through different signal transduction pathways, our results way suggest that certain allergen may selectively alter cytokine release from KU812 cells via different cellular mechanisms.Similarly, it was observed that IL-31, IL-33, IL-37, TSLPstimulated IL-6 release, but not IL-13 release from KU812 cells, which implicates that these cytokines are able to selectively activate IL-6 release signaling pathway.Since IL-13 is a classical Th2 cytokine, and allergens can induce IL-6 and IL-13 release from KU812 cells, it is possible that allergens may contribute to allergy via upregulation of expression of TLR7 and enhancement of IL-6 or IL-13 production in basophils.The ndings that birch pollen allergen stimulated PBMC cultures from asthmatic patientsproduced elevated levels of IL-5 and IL-13,Dermatophagoides farinae or house duststimulated increase in IL-13 production from PBMC culturesfrom patients with atopic dermatitis, and Der p extracts, TNF-α, IL-4, or IL-13 enhanced GM-CSF and IL-8 release from airway epithelial cultures may emphasize the effect of allergens on the release of Th2 cytokines.

Conclusion
In conclusion, the percentages of TLR7 + basophils and TLR7 expression intensity in a single basophil are both increased in blood of the patients with AR, indicating that basophils likely contribute to the pathogenesis of AR via TLR7.The enhanced expression of TLR7 on KU812 cells, induced by allergens Der p1 and OVA at both protein and mRNA levels suggested that allergens may be capable of upregulating TLR7 expression on basophils.Flow cytometry analysis of expression of TLR7 in KU812 cells.Cells were challenged by various concentrations of OVA (0.03, 0.3, 1.0 and 3.0 g/ml), Der p1 (0.03, 0.3, 1.0 and 3.0 g/ml), DSR6434 (0.001, 0.01, 0.1 g/ml), IL-31 at 3 ng/ml, IL-33 at 3 ng/ml, IL-37 at 3 ng/ml, TSLP at 3 ng/ml, plasma from healthy control (HC, 10%) subjects, House Dust Mite extract (HDME, 3 μg/ml), plasma from allergic rhinitis (AR) sensitive to mite alone (10%), Artemisia sieversiana wild allergen extract (ASWAE, 3 μg/ml), plasma from seasonal AR sensitive to artemisia alone (10%) at 37℃ for 2 h (A) and 16 h (B) before cells being collected.The expression of TLR7 was analyzed by ow cytometry analysis.The data were expressed as mean ± SE for ve separate experiments performed in duplicate.P < 0.05 compared with the response to corresponding medium alone control.

Figures Figure 1 Flow
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