A 7-Amino Acid Peptide Mimic from Hepatitis C Virus Hypervariable Region 1 Inhibits Mouse Lung Th9 Cell Differentiation by Blocking CD81 Signaling during Allergic Lung Inflammation

T helper (Th) cells orchestrate allergic lung inflammation in asthma pathogenesis. Th9 is a novel Th cell subset that mainly produces IL-9, a potent proinflammatory cytokine in asthma. A 7-amino acid peptide (7P) of the hypervariable region 1 (HVR1) of hepatitis C virus has been identified as an important regulator in the type 2 cytokine (IL-4, IL-5, and IL-13) immune response. However, it is unknown whether 7P regulates Th9 cell differentiation during ovalbumin- (OVA-) induced allergic lung inflammation. To address this, we studied wild-type mice treated with 7P and a control peptide in an in vivo mouse model of OVA-induced allergic inflammation and an in vitro cell model of Th9 differentiation, using flow cytometry, cytokine assays, and quantitative PCR. The binding of 7P to CD81 on naïve CD4+ T cells during lung Th9 differentiation was determined using CD81 overexpression and siRNA knockdown analyses. Administration of 7P significantly reduced Th9 cell differentiation after OVA sensitization and exposure. 7P also inhibited Th9 cell differentiation from naïve and memory CD4+ T cells in vitro. Furthermore, 7P inhibited the differentiation of human Th9 cells with high CD81 expression from naïve CD4+ T cells by blocking CD81 signaling. CD81 siRNA significantly reduced Th9 cell differentiation from naïve CD4+ T cells in vitro. Interestingly, CD81 overexpression in human naïve CD4+ T cells also enhanced Th9 development in vitro. These data indicate that 7P may be a good candidate for reducing IL-9 production in asthma.


Introduction
Asthma is a chronic airway disease characterized by inflammation, airway hyperreactivity (AHR), and reversible airway obstruction [1]. Complicated innate and adaptive immune responses play significant roles in airway inflammation. Allergen-specific T helper (Th) 2 cells produce key cytokines (IL-4, IL-5, IL-9, and IL-13) for this process [2]. Th9 cells were first identified as a Th2 subpopulation that produced exceptionally large quantities of cytokine IL-9. IL-9 has various effects on numerous hematopoietic cells, which are central to asthma pathogenesis. IL-9 enhances T and mast cell (MC) proliferation and differentiation and promotes IgE production by B cells [3]. The association of IL-9 with susceptibility to developing AHR has been confirmed via human and murine studies [4]. Furthermore, IL-9 elicits eosinophilic and lymphocyte inflammation, mucus production, AHR, and subepithelial collagen deposition in the lungs of mice. The use of neutralizing IL-9 antibodies has been shown to reduce inflammation and AHR. Studies using IL-9-deficient mice demonstrated the redundant role of this cytokine in a similar model of asthma [5]. Mapping studies in mice and humans have shown that IL-9 is an asthma-related gene [6], and anti-IL-9 antibodies have been trialed to treat asthma patients in a clinical setting [7].
The hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) has been identified as an important regulatory factor in the type 2 cytokine (IL-4, IL-5, and IL-13) immune response [8][9][10][11]. Therefore, some mimic peptides derived from HVR1 of HCV were investigated for their roles in the immune response. A designated 7-amino acid peptide (7P; GQTYTSG) was identified by its ability to induce an interesting cytokine profile. The expression levels of IFN-γ, IL-10, and TNF-α in PBMC of HCV patients could be regulated by the 7P stimulation [12,13]. However, it was unknown whether 7P could regulate lung Th9 differentiation, inflammatory responses, and AHR during ovalbumin (OVA)induced allergic lung inflammation. It has been reported that HVR1 of HCV interacted with hepatocytes, CD4 + T cells, and CD8 + T cells through surface CD81 molecules [14]. CD81 is a widely expressed cell-surface protein and is associated with a variety of biological responses in the immune system [15]. More studies have confirmed that CD81 is associated with T and B cell differentiation and expression [16,17]. Furthermore, CD81 is related to MHC class II molecules, integrins, and other tetraspanins [18]. Studies also suggest that CD81 is involved in cell motility, adhesion, proliferation, and differentiation. CD81-deficient (CD81 −/− ) mice and chimeric mice, in which only B cells lack CD81, have been demonstrated to have impaired humoral immune responses to protein antigens (Ags) [19] and Th2 responses [20]. Furthermore, allergen-induced AHR is diminished in CD81 -/mice through the regulation of Th2 cell differentiation and function [21]. These studies have shown that CD81 is involved in allergen-induced AHR via Th2 regulation. It is still unknown whether CD81 and its ligand are also involved in the regulation of Th9 cells during allergic lung inflammation.
Th9 cells develop in vitro in the presence of IL-4 and transforming growth factor-(TGF-) β and secrete high levels of IL-9, IL-10, CCL17, and CCL22 [22]. However, the transcription factors that regulate IL-9 are not well defined. The transcription factors IRF4 and PU.1 are required for IL-9-secreting T cell development [23]. PU.1 binds directly to the IL-9 promoter and is required for the development of allergic inflammation [24]. Although IRF4 binds to the IL-9 promoter, it also induces GATA3 expression during Th2 differentiation [25]. This function may impact Th9 development.
7P is a mimic peptide derived from the HVR1 of HCV, which has been determined to regulate type 2 cytokines such as IL-4, IL-5, IL-9, and IL-13. Furthermore, IL-9 is an important factor in asthma pathogenesis [13,26,27], and anti-IL-9 antibody has been shown to treat asthma in phase II clinical trials [7]. In this study, we therefore investigated the effect of 7P in regulating lung Th9 cell differentiation during allergic lung inflammation in vivo and in vitro. We compared lung Th9 cell differentiation in 7P and vehicle minipump-treated mice during OVA-induced allergic lung inflammation in vivo.

Materials and Methods
2.1. Animals. Male 6-10-week-old C57BL6J mice (50 mice) were purchased from Nanjing Model Animal Research Center (MARC). All animals were provided with sterilized food and water. The study was approved by the Animal Care and Use Committee of the Nanjing Han & Zaenker Cancer Institute (NHZCI) and performed strictly according to the Guide for the Care and Use of Laboratory Animals.

OVA-Induced Allergic Airway Inflammation Model.
Mice were immunized with OVA (20 μg emulsified in 0.2 mL aluminum hydroxide adjuvant) or vehicle (adjuvant only) by intraperitoneal injection on days 0 and 1. Starting on day 14, mice were exposed to 1% OVA or saline via nebulizer for 30 min per day for four consecutive days. Fortyeight hours after the last exposure, mice were euthanized, and tissues were collected.
2.5. Dendritic Cell (DC) Isolation. Mouse spleens were placed into single-cell suspensions with a 2 mL syringe. Spleen DCs were enriched from spleen single-cell suspension with an EasySep™ Mouse Pan-DC Enrichment Kit (STEMCELL, Cat: #19763). CD11c + DCs were further sorted with antimouse CD11c-PE antibodies from the enriched lung DCs by BD FACSAria II.

Implantation of Osmotic Minipumps for Delivery of 7P
and Sham-7P. 7P and sham-7P were dissolved in a vehicle containing 15% ethanol/sterile saline. These were delivered 1 week prior to OVA exposure via subcutaneously implanted osmotic minipumps (Alzet, Cupertino, CA, model 1004). Minipumps were filled with 100 μL of either 6 μg/mL 7P or sham-7P. With an average release rate of 0.11 μL/h, this provided a final delivery rate of 1.32 μg/h for 7P and sham-7P.

Flow Cytometry Analysis and Intracellular Cytokine
Staining. After stimulation of Th9 differentiation from naïve CD4 + T cells with TGF-β and IL-4, cells were incubated for 4 h with 500 ng/mL 12-O-tetradecanoylphorbol-13-acetate, 500 ng/mL ionomycin (Sigma-Aldrich), and 1 μg/mL brefeldin A (GolgiPlug; BD). Cells were fixed and permeabilized using BD Cytofix/Cytoperm™ solution after surface staining. Phycoerythrin-(PE-) conjugated anti-IL-9 mAb (BioLegend) was used to detect intracellular IL-9 and IL-10 levels. Naïve CD4 + T cells and Th9 cells were stained with the conjugated antibodies for 10 min. Cells stained with the appropriate antibodies were analyzed using an LSRII flow cytometer (Becton Dickinson). The data were collected and analyzed using FACSDiva software.
2.9. RNA Isolation and Real-Time PCR. Total RNA was isolated using an RNeasy mini kit (Qiagen), and cDNA was synthesized with a High-Capacity cDNA Archive Kit (Applied Biosystems). TaqMan primer/probe sets for mouse GAPDH (Mm99999915_m1), IL-9 (Mm00434305_m1), IL-10 (Mm00439614_m1), and IRF4 (Mm00516431_m1) were purchased from Applied Biosystems. Samples were analyzed using an ABI Prism 7700 Thermocycler (Applied Biosystems), and differential expression was calculated using the ΔΔCT method.
2.11. siRNA Knockdown of CD81 Receptors. Multiple MIS-SION siRNAs for each receptor were purchased from Sigma-Aldrich for knockdown analysis (Human CD81: SASI_Hs01_00181702; SASI_Hs01_00181703; SASI_Hs01_ 001817024). Naïve CD4 + T cells freshly isolated from the spleens of wild-type mice were transfected with CD81specific siRNAs (20 nmol) or control siRNA using Mouse T Cell Nucleofector Solution (Amaxa). Transfected cells were differentiated into Th9 cells and analyzed via FACS on day 5.

Histopathological
Staining of Lung Tissue. Lungs were perfusion-fixed, and adjacent sections were stained with hematoxylin and eosin (H&E). Pathological evaluation was performed by a pathologist.
2.13. Enzyme-Linked Immunosorbent Assay (ELISA). BALF samples were also taken to measure IL-5, IL-9, and IL13 production. Briefly, BALF samples were collected in 2 mL PBS. An ELISA kit (eBioscience) was used to observe levels of IL-5, IL-9, and IL-13 according to the manufacturer's instructions.
2.14. Statistical Analysis. Data are represented as the mean ± standard error of the mean ðSEMÞ. Statistical comparisons were performed by randomized-design two-way analysis of variance (ANOVA), followed by the Newman-Keuls post hoc test for more than two groups or unpaired Student's t-test for two groups. Statistical significance was defined as p < 0:05.

7P Dramatically Suppresses Th9 Differentiation from
Both Naïve CD4 + T Cells and OVA-Memory CD4 + T Cells In Vitro. To examine the mechanism by which 7P inhibits lung Th9 cell differentiation during allergic lung inflammation, we applied an in vitro Th9 differentiation method from naïve CD4+ T cells to observe the effect of 7P on suppressing Th9 cell differentiation. Briefly, we treated naïve CD4 + T cells isolated from mouse spleens with anti-CD3, anti-CD28, and Th9-inducing cytokines (TGF-β and IL-4) to induce Th9 cell differentiation in the presence and absence of 7P, sham-7P, and vehicle (Figure 2(a)). Il9, Il10, and Irf4 expressions were investigated to identify Th9 cell differentiation. The results   Journal of Immunology Research indicated that Th9 cell lineage markers (Il9, IL10, and Irf4) were significantly decreased following treatment of naïve CD4 + T cells with TGF-β, IL-4, and 7P (Figures 2(b)-2(d)). Human Th9 differentiation from human peripheral naïve CD4 + T cells was significantly inhibited by 7P (Figure 2(e)).
These data indicate that 7P is a negative regulator of Th9 cell differentiation from naïve CD4 + T cells and confirms that IL-9 production is decreased with 7P treatment. Although 7P has been observed to inhibit Th9 cell differentiation from naïve CD4 + T cells in vitro, it is unknown whether 7P also inhibits Th9 cell differentiation from OVA-memory CD4 + T cells. To address this issue, we immunized mice with OVA for 14 days, followed by isolating OVA-memorized CD4 + T cells (CD3 + /CD4 + /CD62L -/CD44 high+ T cells). We then restimulated them with OVA in vitro for 5 days (Figure 3(a)). The OVA-restimulated memory CD4 + T cells were collected, and RNA was isolated. Il9, Il10, and Irf4 mRNA were quantified by qPCR. The results show that Il9, Il10, and Irf4 mRNA were significantly decreased in the 7Ptreated group compared with the vehicle and sham-7P groups (Figures 3(b)-3(d)). These data demonstrate that 7P not only suppresses Th9 cell differentiation from naïve CD4 + T cells but also inhibits Th9 cell differentiation from memory CD4 + T cells.

Th9 Cells Have High CD81 Expression and Can Bind 7P.
It is established that CD81 is expressed in T cells and regulates the differentiation and function of Th1 and Th2 cells [28]. However, it was unknown whether Th9 cells express CD81. Previous studies have demonstrated that CD81

Journal of Immunology Research
signaling plays an important role in promoting Th2 cells in asthma [29]. Therefore, blocking CD81 signaling may inhibit Th cell differentiation. To define the mechanism by which 7P inhibits Th9 cell differentiation, we firstly examined whether Th9 cells express CD81 during mouse and human Th9 cell differentiation. This was determined in vitro by qPCR and flow cytometry. The results indicated that levels of CD81 mRNA (Figure 4(a)) and protein expression (Figure 4(b)) by mouse Th9 cells were significantly increased during Th9 cell differentiation from naïve CD4 + T cells compared with the vehicle. Consistent with CD81 expression observed in mouse Th9 cells, expression levels of CD81 mRNA and protein by human Th9 cells were also increased, indicating that human Th9 cells had increased CD81 expression (Figures 4(c) and 4(d)). Secondly, to examine whether 7P binds to CD81, we incubated 7P conjugated with FITC (7P-FITC) with CD4 + T cells (Jurkat cells) transfected with pcDNA3.1-CD81 or pcDNA3.1 vector plasmids. High CD81 expression was confirmed by flow cytometry in CD4 + T cells transfected with pcDNA3.1-CD81 (Supplementary Figure 2B). The results indicated that the CD4 + T cells with higher CD81 expression (transfected with pcDNA3.1-CD81) were able to bind a high density of 7P-FITC. In contrast, CD4 + T cells with lower CD81 expression (transfected with pcDNA3.1 only) were able to bind a low density of 7P-FITC. These results demonstrate that CD81 is significantly upregulated during Th9 cell differentiation, and 7P binds to CD81 in vitro (Figures 4(e) and 4(f)).

7P Blocks CD81 Signaling to Inhibit Th9 Cell Differentiation In Vitro and In Vivo.
To determine the effect of 7P-CD81 signaling in regulating Th9 cell differentiation, we examined Il9 and Irf4 mRNA levels during Th9 cell differentiation in vitro after Cd81 knockdown by siRNAs in Th9 cells. Cd81-siRNA1 significantly inhibited Cd81 mRNA expression by Cd81-siRNA-1 (Figure 5(a)). The expression of Il9 and Irf4 mRNA was markedly lower in Th9 cells differentiated from Cd81-siRNA1-transfected naïve CD4 + T cells compared to those transfected with Sham-siRNA and Cd81-siRNA3 (Figures 5(b) and 5(c)). The results suggested that the CD81 signaling pathway promoted Th9 cell differentiation from naïve CD4 + T cells.
To further confirm the effect of 7P on CD81 signaling in Th9 differentiation, we constructed a pcDNA3.1-CD81 expression plasmid (Supplementary Figure 2). We transfected this plasmid into human Jurkat lymphocyte cells and further treated CD81-overexpressed Jurkat cells, normal Jurkat cells, human naïve CD4 + T cells, and human naïve CD4 + T cells with TGF-β plus IL-4-and CD81-activated antibody for 5 days. This was done with or without 7P blocking, and the development of the Th9 cell phenotype was examined. The results indicated that CD81-overexpressed cells produced higher levels of Il9 and Irf4 mRNA than normal Jurkat cells ( Figure 5(d)) and human naïve CD4 + T cells ( Figure 5(f)). 7P significantly blocked the Il9 and Irf4 mRNA transcripts promoted by anti-CD81 antibody in CD81-overexpressed human naïve CD4 + T cells and Jurkat cells. IL-9 + /CD4 + T cell analysis via FACS also obtained results consistent with this information (Figures 5(e) and 5(g)). These data indicated that 7P blocked CD81 signaling, thus decreasing Th9 cell differentiation.

Discussion
CD81-deficient mice have been known to exhibit impaired Th2 cell responses, reduced AHR, and decreased airway inflammation. This occurs by influencing Ag-specific IgE production and regulating local cytokine production [30].    Journal of Immunology Research In this study, we reported that 7P blocked CD81 signaling, significantly reducing lung Th9 cell differentiation during allergic lung inflammation. Our novel findings include (1) 7P significantly suppressed allergic lung inflammation and lung Th9 cell differentiation during OVA-induced allergic lung inflammation in vivo; (2) 7P not only decreased Th9 cell differentiation from naïve CD4 + T cells but also inhibited Th9 cell development from memory CD4 + T cells in vitro; (3) 7P could bind to CD81, which was highly expressed in Th9 cells, and CD81 signaling promoted Th9 cell development in vitro; and (4) 7P blocked CD81 signaling to reduce Th9 cell differentiation after OVA sensitization/exposure. Th9 cells are a unique subset of effector T cells distinct from the Th1, Th2, and Th17 cell subsets. Th9 cells secrete IL-9, IL-10, and other cytokines, which play essential roles in human asthma pathogenesis [31,32]. In this study, we found that 7P significantly decreased allergic lung inflammation and lung Th9 cell differentiation during OVA-induced allergic lung inflammation. Based on this, we determined that 7P is a critical negative regulator of Th9 cell differentiation and IL-9 production during allergic lung inflammation. 7P significantly decreased this process, as well as suppressing lung Th9 cell differentiation during OVA-induced allergic lung inflammation in vivo. The results suggested that IL-9secreting Th9 cells play a critical role in allergic lung inflammation. Therefore, inhibiting IL-9 production may be another potent avenue for treating asthma. Recently, the anti-IL-9 antibody has been used to treat asthma patients in the clinical trial (phase II) at the NIH [33,34]. 7P could dramatically suppress Th9 cell differentiation, suggesting that 7P may be a good candidate for reducing IL-9 production in asthma.
Our findings determined that 7P bound to CD81 and blocked the CD81 receptor, inhibiting lung Th9 cell differentiation. These results present data that contradicts previous results, which have indicated that HVR1 does not bind to CD81 and that HVR2 does so [35]. As 7P is a mimic peptide derived from the HVR1 of HCV, we questioned the reason why 7P bound to CD81 on Th9 cells in our results. Although a study has reported that HVR1 could not bind to CD81, it has been demonstrated that HVR of HCV interacts with hepatocytes and CD4 + and CD8 + T cells through surface CD81 molecules [14,15]. It was therefore difficult to determine whether HVR1 of HCV binds to CD81. Our data here provides strong evidence to support that 7P could bind to CD81 in Th9 cells. CD81 is a widely expressed cell-surface protein involved in a variety of biological responses, mostly studied in the context of the immune system [36]. CD81 is also an important factor in airway inflammation and Th9 cell differentiation during allergic lung inflammation. CD81 and CD28 co-stimulate T cells through a distinct pathway [37], and CD81 may result in the activation of TCRγδ T cells [38]. Human CD81 directly enhances Th1 and Th2 cell activation but preferentially induces Th2 cell proliferation upon long-term stimulation [39]. The presence of CD81 on B cells promotes IL-4 secretion and antibody production during the Th2 immune response [29]. It was reported that CD81deficient mice had reduced airway inflammation, AHR, and Th2 cell differentiation in allergic lung inflammation [40]. CD81 expression in CD4 + T cells is induced by Th9-inducing factors during asthma attacks. CD81 signaling causes several proinflammatory effects in the lungs, including bronchoconstriction and eosinophilia. In contrast, 7P has a protective effect in models of allergic inflammation by suppressing bronchoconstriction and Th9 cell proliferation via blocking CD81 signaling. Previous data have shown that CD81 promotes Ag-induced lung inflammation and that 7P has a protective role in allergic lung inflammation. Consistent with these findings, we found that 7P significantly reduced Th9 cell differentiation in mice. Blocking CD81 signals leads to decreased Th9 cell differentiation and IL-9 secretion, which may then contribute to the decreased lung inflammation observed in 7P-treated mice. More importantly, this result is consistent with the previously known inhibitory role of HVR1 in IL-9 expression [12].
Our study further defined the precise molecular mechanisms underlying the regulation of Th9 cell differentiation by 7P-CD81 signaling. CD81 receptor knockdown in naïve CD4 + T cells by siRNA significantly decreased Th9 cell differentiation. The role of CD81 was further confirmed using CD81-overexpressing human CD4 + T cells. Our results suggest that 7P plays a vital role in regulating Th9 cell differentiation by altering CD81-receptor signals during allergic lung inflammation. Furthermore, we determined that 7P also controlled the differentiation of human T cells to Th9 cells. Similar to observations found in murine studies, 7P diminished the differentiation of human naïve CD4 + T cells in peripheral blood to Th9 cells, further indicating that 7P may be a good candidate for reducing IL-9 production in asthma.

Conclusions
We have provided in vitro and in vivo evidence to demonstrate that 7P inhibits Th9 cell differentiation and allergic lung inflammation by blocking CD81 signaling, thus downregulating Th9 differentiation. Our results demonstrate the important role of 7P-CD81 signaling in regulating Th9 cell differentiation.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no competing financial interests.