TGFβ1 mimetic peptide modulates immune response to grass pollen allergens in mice

Abstract Background Transforming growth factor β1 (TGFβ1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFβ1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFβ1 mimetic peptide (TGFβ1‐mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. Methods The in vitro action of TGFβ1‐mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL‐4 reporter mice. Results In vitro, TGFβ1‐mim downregulated TNF‐α production, IL‐8 gene expression, and cytokine secretion, upregulated IL‐10 secretion, and inhibited Phl p 5‐induced basophil degranulation. During Phl p 5 sensitization in mice, TGFβ1‐mim downregulated IL‐2, IL‐4, IL‐5, IL‐13, and IFN‐γ, upregulated IL‐10, and induced Treg cell production. Furthermore, mice treated with TGFβ1‐mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. Conclusion The TGFβ1‐mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen‐specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.


| INTRODUC TI ON
Allergens are environmental proteins that interact with innate immune receptors, leading to Th2 polarization and consequently IgE production. 1 Grasses are among the most potent sources of allergens. They produce large amounts of pollen, are widely distributed, and are potent respiratory allergens. Although it is estimated that only 8% of grass pollen-allergic patients have been officially diagnosed, it is believed that around 20% of the population of the United States, Europe, and Brazil are affected by grass pollen allergy. 2,3 Hence, grass pollen allergy is a global problem, implying a need for novel therapeutic approaches. Almost all timothy grass (Phleum pratense) allergic patients displaying strong clinical reactions have high IgE antibody responses toward Phl p 5. [4][5][6] Allergic sensitization is initiated when an allergen interacts with innate immune receptors, leading to Th2 polarization and IgE production. Upon re-exposure, allergen cross-links IgE bound to the high-affinity FcεRI receptor on mast cells leading to inflammatory reactions characterized by the secretion of chemical mediators, synthesis of leukotrienes, prostaglandins, and cytokines (eg, IL-4, IL-5, and IL- 13), and by recruitment of other effector cells, such as Th17, Th9, basophils, and eosinophils. [7][8][9] Th1 cells also contribute to the effector phase and chronicity in allergic diseases.
Regulatory T (Treg) cells are essential for immune tolerance against autoimmune and inflammatory diseases, allergies, and various other events related to the breakdown of immune homeostasis. Treg cells are a heterogeneous population characterized by the constitutive expression of the transcription factor Foxp3 and represent the dominant subset specific for common environmental allergens in healthy individuals. [10][11][12] Transforming growth factor β1 (TGFβ1) is a key cytokine involved in the induction of Treg cells and in the regulation of effector T cells, B cells, and epithelial cells. 13 In mice possessing a disrupted TGFβ1 gene or lacking the TGFβ receptor II (TGFβRII), severe inflammatory responses, tissue necrosis, and early death were observed, 14,15 confirming the broad immune regulatory functions of TGFβ1. Hence, the modulation of allergen-specific CD4 + effector T cells by TGFβ1 could potentially induce immune tolerance and suppress allergic inflammation.
Considering that the availability, specificity, and biological activity of TGFβ1 are normally controlled by numerous interactions with membrane-bound proteins, 16 short peptides that mimic the binding domain of TGFβ1 and are able to efficiently activate the TGFβRII on target cells might represent novel therapeutic tools for treating allergies. We have previously selected by phage display a TGFβ1 mimetic peptide (pm26TGFβ1/TGFβ1-mim) that was mapped into the TGFβ1:TGFβRII activation domain and shown to contain amino acid residues that are crucial for high-affinity binding and stabilization. Furthermore, in the same study we also showed that the TGFβ1-mim displayed potent anti-inflammatory activity. 17 Here, we investigated the ability of the TGFβ1-mim to modulate the allergic/inflammatory response in vitro and in mice sensitized with Phl p 5 or timothy grass pollen extract. Our findings showed that the TGFβ1-mim peptide efficiently modulated crucial events involved in allergen-specific immune responses.

| Synthetic peptide
LPS-free TGFβ1-mim (pm26TGFβ1) 17 was chemically synthesized by Bachem (Weil am Rhein, Germany). A detailed description is G R A P H I C A L A B S T R A C T TGFβ1-mim downregulated major cytokines involved in Th1 and Th2 responses in mice sensitized to Phl p 5. Treatment of mice with the mimetic during sensitization to Phl p 5 induced Treg production, suppressed specific IgE and IgG1, IgE-mediated basophil degranulation, induced IgA antibodies and IL-10 secretion. In an IL-4 reporter model, TGFβ1-mim suppressed Th2 polarization induced by grass pollen extract. Abbreviation: TGFβ1-mim, TGFβ1 mimetic peptide provided in the Appendix S1. Unconjugated human TGFβ1 recombinant protein produced in CHO cells (eBioscience, Austria) was included as positive control in the in vitro experiments.

| IL-8 expression analysis
To investigate whether TGFβ1-mim affected IL-8 expression, an A549 immortalized human lung epithelial cell line (A549 -ATCC, LGC Promochem, Wesel, Germany) stably transfected with a luciferase reporter gene placed under the control of the IL-8 promoter was used according to the original protocol. 18 A detailed description is provided in the Appendix S1.

| IL-10 and TNF-α measurements
Levels of secreted IL-10 and TNF-α in the supernatant of Jurkat cells and IL-8 in the supernatant of A549 cells were measured by ELISA.

| In vivo Th2 polarization model
Bicistronic IL-4 reporter mice in the BALB/c background (8-13 weeks old) were obtained from The Jackson Laboratory.
Because our preliminary data showed that rPhl p 5 alone did not induce IL-4 production, we investigated the Th2 polarizing capacity of grass pollen extract alone or in combination with the mimetic.

| Rat basophil leukemia (RBL) cell mediator release assay
The capacity of TGFβ1-mim to inhibit Phl p 5-induced degranulation in murine RBL-2H3 (muRBL) cells (ATCC ® CRL2256™) was performed as described elsewhere. 21 Humanized RBL-2H3 (huRBL) cells transfected with the cDNA encoding the human FcɛRI were tested with nine sera from grass pollen-allergic patients, as described elsewhere. 22 Experiments using anonymized human serum samples were approved by the local Ethics Committee of the Medical University and General Hospital of Vienna (no. EK1263/2014), and informed written consent was obtained from all study participants. A detailed description is provided in the Appendix S1.

| Phl p 5-specific antibody detection by ELISA
The levels of Phl p 5-specific IgE, IgG1, IgG2a, and IgA antibodies were measured by ELISA in sera from all groups of mice. A detailed description is provided in the Appendix S1.

| Cytokine-producing cells detection by ELISPOT
Production of IFN-γ, IL-4, and IL-10 from splenocytes in response to Phl p 5 stimulation was assessed by ELISPOT. A detailed description is provided in the Appendix S1.

| Flow cytometry analysis
Flow cytometry analyses of proliferating mice splenocytes upon Phl p 5 stimulation for 5 days were performed as described in the Appendix S1. In this study, we investigated the percentage of allergen-specific CD4 + Foxp3 + Treg cells expressing CD25, GATA3, CTLA4, or the cellular proliferation marker KI67, important factors associated to Treg cell function.

| Statistical analysis
Statistical analyses were conducted using GraphPad Prism 5 software (GraphPad software). Results are presented as mean with SD of each group. Comparisons between groups were performed using one-way ANOVA for all experiments, except for the human RBL assays where the paired Student's t test was employed. P values < .05 (*P < .05; **P < .01; ***P < .001) were considered significant.

| TGFβ1-mim modulates cytokine production and IgE-mediated basophil degranulation in vitro
We first tested the ability of the TGFβ1-mim peptide to recognize TGFβRII on Jurkat cells, using ELISA. Recombinant TGFβ1 was used as a positive control. Although not statistically significant, TGFβ1mim showed slightly elevated reactivity to TGFβRII than rTGFβ1 ( Figure 1A). In PMA-stimulated Jurkat cells, both mimetic and rTGFβ1 significantly decreased the secretion of TNF-α ( Figure 1B) and increased the secretion of IL-10 ( Figure 1C). Since the lung epithelium plays an important role as a first line of defense toward external compounds, 18 we sought to investigate the production of IL-8 using a human lung epithelial (A549) cell line possessing a luciferase reporter gene under the control of the IL-8 promoter. In TNF-α-stimulated A549 cells, both TGFβ1-mim and rTGFβ1 decreased IL-8 gene expression ( Figure 1D), whereas only TGFβ1-mim significantly decreased IL-8 secretion ( Figure 1E). To investigate whether the mimetic modulates IgE-mediated basophil degranulation in an already established allergic microenvironment, we performed mediator release assays using RBL cells expressing the humanized FcεRI receptor. We found that the amount of Phl p 5 needed to induce half-maximal release in basophils, which were passively sensitized with sera from grass pollen-allergic patients, was significantly higher when cells were pretreated with TGFβ1-mim ( Figure 1F), indicating an suppression of degranulation. This effect was also observable when pretreating with rTGFβ1. Titration curves for every patient are presented in Figure E1.
These results indicate that the TGFβ1-mim was able to modulate the allergic inflammatory microenvironment in vitro.

| TGFβ1-mim modulates antibody response in vivo
We next tested the ability of the TGFβ1-mim to modulate the antibody response during Phl p 5 sensitization. Phl p 5 (Figure 2A) significantly induced allergen-specific IgE response, as observed from the capacity of mice sera to provoke degranulation in RBL cells ( Figure   E2 and Figure 2B). RBL cells sensitized with sera of mice treated with 1 µmol/L TGFβ1-mim had significantly lower levels of degranulation than the group injected with Phl p 5 alone. ELISA analysis showed that in serum from Phl p 5-sensitized mice, TGFβ1-mim significantly suppressed the levels of allergen-specific IgE ( Figure 2C) and IgG1 ( Figure 2D). No significant differences were observed for allergenspecific IgG2a antibodies ( Figure 2E). Mice treated with TGFβ1mim also showed significant higher levels of allergen-specific IgA ( Figure 2F). Therefore, TGFβ1-mim modulated the Phl p 5-specific antibody responses in mice.

| TGFβ1-mim modulates cytokine production in vivo
To investigate the cytokine profile in the supernatants of restimulated splenocytes, we used a multiplex cytokine analysis kit. The secretion of IFN-γ, IL-2, IL-4, IL-5, and IL-13 was significantly lower, whereas secretion of IL-10 was higher in mice treated with TGFβ1mim than in untreated mice ( Figure 3A). To determine the antigenspecific T cell polarization upon TGFβ1-mim treatment, the number of IFN-γ, IL-4, and IL-10 producing splenocytes in all groups was assessed by ELISPOT. Sensitization with Phl p 5 resulted in high induction of IFN-γ and IL-4 release upon allergen stimulation. In mice treated with TGFβ1-mim, levels of IFN-γ and IL-4 secreted by Phl p 5-restimulated splenocytes were significantly lower, while levels of IL-10 were significantly higher than in untreated mice ( Figure 3B). Therefore, TGFβ1-mim downregulated Th1 and Th2 cytokines, and upregulated IL-10.
These results demonstrate that TGFβ1-mim promotes Treg cell production during Phl p 5 sensitization.

| TGFβ1-mim suppresses Th2 polarization in vivo
Th2 lymphocytes produce the cytokines IL-4, IL-5, and IL-13, and drive the class switching toward IgE in B cells. 25 To investigate whether the TGFβ1-mim modulated Th2 polarization in the pres-    has been shown to inhibit IL-8 production. 40,41 In our murine in vivo model, sensitization was induced by Phl p 5. The pretreatment with TGFβ1-mim followed by Phl p 5 sensitization resulted in the modulation of various events related to the exacerbation of the allergic response. Firstly, in vivo treatment with TGFβ1-mim significantly inhibited antigen-specific IgE and IgG1 antibody production and suppressed degranulation in basophils passively sensitized with sera from Phl p 5-sensitized mice. TGFβ1-mim enhanced IgA production, consistent with other studies reporting TGFβ1 as an IgA-specific class-switching factor. [42][43][44] Furthermore, allergic disorders appear to be more common among patients with deficiency for IgA. 45, 46 We also observed that sera from mice treated with TGFβ1-mim had enhanced IgE-allergen blocking capacity (data not shown), which will be further investigated in future studies. In order to investigate the modulatory capacity of the TGFβ1-mim on Th2 polarization in vivo, we used the 4get mouse model, which possesses a bicistronic mRNA linking a readily identifiable reporter (eGFP) to the IL-4 gene expression. 26,27,61,62 In this model, recombinant Phl p 5 alone was not able to induce expression of eGFP in CD4 + T cells. In a recent study, we could show that birch pollen extracts promoted Th2 polarization in the 4get mouse model whereas purified recombinant Bet v 1 did not. These findings led us to hypothesize that the context in which an allergen is presented to the immune system plays a crucial role in the outcome of the allergen-specific immune response. 27 In line with these observations, here we showed that grass pollen extract induced Th2 polarization in the 4get mice but not rPhl p 5, thus reinforcing the idea that the context (adjuvants) provided by the pollen is crucial in the initiation of the Th2 polarization.

| D ISCUSS I ON
TGF-β1 has been implicated in the pathogenesis of human fibrosis. 63 Additionally, several cytokines including IFN-γ, IL-4, and IL-13 have been shown to participate in the progression of fibrosis. [64][65][66] A crucial role of toll-like receptor 4 (TLR4) in fibrosis has also been suggested. 67 Although the implication of the TGFβ1-mim in the induction of fibrosis was not directly addressed in this study, we found that under in vivo inflammatory conditions induced by allergen sensitization, the mimetic was able to efficiently downregulate the production of IFN-γ, IL-4, and IL-13 ( Figure 3). We also observed that the mimetic was able to downregulate the LPS-induced TLR4 expression in a human TL4 reporter cell line (data not shown). In this respect, overexpression of TLR4 after LPS challenge in mice or mouse lung fibroblasts was shown to significantly contribute to the induction of pulmonary fibrosis. 68 Future studies should explore the immunomodulatory potential of the TGFβ1-mim within other models of allergic sensitization (eg, food and house dust mite) as well as different routes of application (eg, mucosal and transdermal).

ACK N OWLED G M ENTS
The work was support by grants from the Brazilian funding agency Galber R. Araujo is a recipient of the European Academy of Allergy and F I G U R E 5 TGFβ1-mim peptide inhibits Th2 polarization in IL-4 reporter mouse model. (A) 4get mice were sacrificed 5 days after allergen injection to analyze the frequency of CD4+ EGFP+ expressing skindraining inguinal lymph node cells.