Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice

Abstract Helminths and their products can shape immune responses by modulating immune cells, which are dysfunctional in inflammatory diseases such as asthma. We previously identified SJMHE1, a small molecule peptide from the HSP60 protein of Schistosoma japonicum. SJMHE1 can inhibit delayed‐type hypersensitivity and collagen‐induced arthritis in mice. In the present study, we evaluated this peptide's potential intervention effect and mechanism on ovalbumin‐induced asthma in mice. SJMHE1 treatment suppressed airway inflammation in allergic mice, decreased the infiltrating inflammatory cells in the lungs and bronchoalveolar lavage fluid, modulated the production of pro‐inflammatory and anti‐inflammatory cytokines in the splenocytes and lungs of allergic mice, reduced the percentage of Th2 cells and increased the proportion of Th1 and regulatory T cells (Tregs). At the same time, Foxp3 and T‐bet expression increased, and GATA3 and RORγt decreased in the lungs of allergic mice. We proved that SJMHE1 can interrupt the development of asthma by diminishing airway inflammation in mice. The down‐regulation of Th2 response and the up‐regulation of Th1 and Tregs response may contribute to the protection induced by SJMHE1 in allergic mice. SJMHE1 can serve as a novel therapy for asthma and other allergic or inflammatory diseases.


| INTRODUC TI ON
Asthma is a chronic inflammatory airway disease. A variety of immune cells and inflammatory mediators (such as eosinophils and Th2 and Th17 cells) and many inflammatory cytokines and chemokines are involved in the airway inflammation of asthma. 1 Although the pathogenesis of asthma remains unclear, CD4 T cells, especially the imbalance of Th1/Th2 and Th17/regulatory T cells (Tregs), affect the inflammatory responses in asthma. 2,3 Although corticosteroids can effectively treat the disease, many patients do not respond to the management and suffer from severe long-term side effects. [4][5][6] Thus, new therapeutic targets on asthma should be urgently developed.
Research has identified the protective effects of some helminth infections against allergic diseases such as asthma. 7 Although helminth infections and allergic diseases have similar immune responses, such as high levels of Th2 cytokines, IgE and eosinophilia, the morbidity of asthma in the epidemic areas of parasitic diseases is low. 8 Helminth infections, such as those caused by Heligmosomoides polygyrus, Schistosoma mansoni and Litomosoides sigmodontis, can modulate airway inflammation in mice. [9][10][11] Furthermore, the prevalence and severity of asthma in individuals present a decrease in the epidemic area of schistosomiasis. 10 Schistosoma infection can suppress the development of allergen-induced airway inflammation in asthmatic mice. 11,12 Helminth infection and helminth-derived components can achieve the same effect. In addition to the therapeutic potential of helminths and their products, the immunomodulation induced by helminths may contribute to the identification of the key regulators of pathogenesis in diseases. Schistosoma antigens modulate airway inflammation via IL-10 and/or CD4 + CD25 + Foxp3 + Tregs in mice 11,13 and change cytokine secretion and the activation of lymphocytes from asthmatic patients. 14 However, infectious or whole proteins may elicit side effects to patients. Therefore, seeking small molecules from helminths, such as peptides, for use as immunomodulatory drugs, can be a safe choice for preventing and treating asthma.
In our previous study, we showed that SJMHE1, which is an HSP60-derived peptide from Schistosoma japonicum, could increase CD4 + CD25 + Tregs in vitro and in vivo. 15 SJMHE1-or SJMHE1-elicited CD4 + CD25 + T cells suppressed delayed-type hypersensitivity (DTH) in mice. 15,16 Moreover, SJMHE1 alleviated the inflammation of collagen-induced arthritis (CIA) in mice. 17 In the present study, we explored the effects of SJMHE1 on experimental asthma in mice. SJMHE1 treatment significantly decreased the infiltrating inflammatory cells in the airways and regulated the cytokine responses in the splenocytes and lungs of asthmatic mice. SJMHE1 treatment also decreased the population of Th2 cells, along with an increase in Th1 and CD4 + CD25 + Foxp3 + Tregs, which may protect asthmatic mice from airway inflammation.
These results highlight the potential efficacy of SJMHE1 for treating a range of human inflammatory diseases.

| SJMHE1 treatment and induction of experimental asthma
Experimental asthma was induced according to a previous study. 18 Apart from the PBS group, the mice were immunized by three intraperitoneal injections of alum-precipitated antigen (0.2 mL) containing 50 µg of OVA (fraction V; Sigma, Poole, UK) and 2 mg of 10% aluminium hydroxide gel in PBS on days 0, 7 and 14. They were treated with emulsified SJMHE1 (10 µg) or PBS with incomplete Freund's adjuvant (Sigma, Poole, UK) on days 0, 14 and 28. From days 21 to 27, the mice were challenged with aerosolized OVA (2%) or PBS for 30 minutes using a ME-U12 ultrasonic nebulizer (Omron, Tokyo, Japan). They were also grouped on the basis of treatment as follows: PBS group, PBS immunized and challenged; OVA group, OVA immunized and challenged; OVA/PBS group, PBS treated and OVA immunized and challenged; OVA/SJMHE1 group, SJMHE1 treated and OVA immunized and challenged. Finally, on day 35, the mice were anaesthetized and killed to evaluate lung and airway inflammation.

| Analysis of cells in bronchoalveolar lavage fluid (BALF)
The left bronchial tubes of the mice were ligated, and the right lungs were washed twice using sterile PBS (0.5 mL) to collect the BALF.
Then, the BALF was centrifuged. Cell pellets were resuspended in 1 mL PBS, and the total quantity of inflammatory cells was counted using a haemocytometer. For eosinophils count, a smear of the cell pellet of the BALF was prepared and examined by Wright and Giemsa staining.

| Histopathologic analysis
After BALF collection, the left lungs were fixed in 10% formalin and embedded in paraffin. Haematoxylin and eosin (H&E) staining was employed to stain the lung tissue of mice. The inflammation of the lungs in mice and histological analyses were determined as previously described. 19

| Immunohistochemical staining
Briefly, lung tissue sections were subjected to antigen retrieval and then blocked and incubated with 1:150 dilution of monoclonal anti Foxp3 antibody (eBioscience) and 1:300 dilution of anti-rat IgG-horseradish peroxidase thereafter (Servicebio). Images were captured using Olympus BX51 microscope (Olympus). Image-Pro Plus 6.0 software (Media Cybernetics) was used to quantify the mean densities of Foxp3, which was stained brown in pixels at 200 × magnification.

| Serum anti-OVA-specific IgE measurement
The quantification of anti-OVA-specific IgE in the sera of mice was carried out by enzyme-linked immunosorbent assay (ELISA).
Briefly, the ELISA plates (Costar) were coated with 100 µL/well OVA (100 µg/mL) in pH 9.6 carbonate-bicarbonate buffer at 4°C overnight and then blocked for 2 hours with 200 µL/well skimmed milk powder (5%). After washing, the diluted sera were added and incubated for 2 hours at 37°C. After washing, 1:250 dilution of goat anti-mouse IgE (Abcam) was added and incubated for 2 hours at 37°C. After removing the unbound antibodies, HRP-conjugated rabbit anti-goat secondary IgG (1:5000, Multisciences) was incubated for 1 hours at 37°C. The colour reaction was developed by adding 100 µL/well of TMB solution (eBioscience) for 15 minutes and then stopped with 50 µL/well of 2 M sulphuric acid. Finally, it was read at 450 nm in an ELISA reader (Bio-Rad).

| Flow cytometry analysis
Splenocytes from mice were suspended in the presence of PMA/ ionomycin mixture (Phorbol 12-myristate13-acetate, Multisciences) and brefeldin/monensin mixture (Multisciences) for 5 hours to analyse the Th1, Th2 and Th17 cells. Then, the cells were collected and stained with PerCP anti-CD3 mAbs (eBioscience) and FITC anti-CD4 mAbs (eBioscience). After removing the unbound antibodies, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences). Then, they were stained with APC mouse anti-IFN-γ (eBioscience), phycoerythrin (PE) mouse anti-IL-4 (BioLegend) and PE mouse anti-17A (BioLegend) following the manufacturer's instructions. To determine Tregs, we used the Mouse Regulatory T Cell Staining Kit (eBioscience) for analysis as previously described. 15 The samples were analysed using BD FACSCanto flow cytometer (BD Biosciences) and Flowjo Software (Tree Star).

| RNA extraction and quantitative RT-PCR (qRT-PCR)
The extraction of RNA and the reverse transcription from splenocytes and lungs of mice were performed using Prime Script 1st Strand cDNA Synthesis Kit (Takara). A quantitative analysis of the relative mRNA expression of cytokines was performed in the spleen cells and lung tissue by qRT-PCR using All-in-one™ Mix (Genecopoeia). All-in-one™ qPCR primer sets for IFN-γ (cat. no. no. MQP027158) was used as an endogenous control for sample normalization. The parameters for PCR amplification were 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 15 seconds. The relative mRNA expression was calculated with the comparative △Ct method using the formula 2 −△△Ct.

| Western blot analysis
The proteins of the lung tissues in mice were extracted for Western blot analysis as described previously. 20  were used as the primary antibodies. HRP-conjugated anti-rat IgG (Beyotime Biotechnology) was also utilized. ECL chemiluminescence kit was used for chemiluminescent detection followed by image analysis.

| Statistical analyses
Statistical analyses were performed with GraphPad Prism 5.01 (GraphPad Software, 2007). Data were expressed as mean ± standard error of the mean. The groups were compared using one way ANOVA with Tukey Kramer post hoc tests, in which a P-value of less than 0.05 was considered as statistically significant.

| SJMHE1 treatment suppresses OVA-induced inflammation in allergic mice
Mice were sensitized and challenged by OVA to examine the effects of SJMHE1 on asthma. The treatment regimen is illustrated in Figure 1A. As shown in Figure 1B, asthmatic mice displayed intense inflammatory cell infiltration in the peribronchial and perivascular regions of the lungs. Meanwhile, SJMHE1 treatment reduced the cellular infiltration of the lungs. The inflammation index was determined using Underwood's standards as described previously. 19 On the basis of inflammation indexes, the OVA and OVA/PBS groups showed more perivascular eosinophilia, epithelial damage and oedema compared with the PBS control group ( Figure 1B and C).
However, compared with those in the OVA or OVA/PBS group, the inflammation scores in SJMHE1-treated mice were significantly reduced ( Figure 1B and C). Furthermore, SJMHE1 treatment significantly decreased the infiltrating inflammatory cells induced by OVA sensitization and challenge because the OVA or OVA/PBS group had significantly higher cell and eosinophilia numbers in the BALF than in the OVA/SJMHE1 group ( Figure 1D and E). OVA-specific IgE was measured by ELISA. As shown in Figure 1F, the OVA and OVA/PBS groups had significantly higher IgE level than the PBS control group. However, the SJMHE1 treatment group showed no reduction in IgE level relative to the OVA or OVA/PBS group. These results indicate that SJMHE1 treatment can suppress airway inflammation in allergic mice.

| SJMHE1 treatment modulates cytokine expression in the splenocytes and lungs of allergic mice
The elevation of Th2 and Th17 cytokines, such as IL-4 and IL-17, in asthmatic patients may affect the pathogenesis of asthma. 2 Parasitic infections and their products can protect against inflammatory diseases by inducing immunomodulatory cytokines, such as IL-10, TGF-β and IL-35. 21 To investigate the effects of cytokines on SJMHE1 treatment, we tested the expression of cytokines in the splenocytes and lungs of mice. As shown in Figure

| SJMHE1 treatment modulates the proportion of Th1/Th2/Th17/Treg subsets in splenocytes of allergic mice
An imbalance of Th1/Th2 and Th17/Treg has been observed in allergic patients. 3 Furthermore, our previous study demonstrated that SJMHE1 treatment can induce CD4 + CD25 + Tregs to suppress DTH responses 16 and alleviate CIA in mice. 17 In the present study, whether SJMHE1 suppresses airway inflammation and modulates cytokine production in allergic mice by regulating the Th subsets

| SJMHE1 treatment increases the expression of Foxp3 in the splenocytes and lungs of allergic mice
Foxp3 is the lineage-specific transcription factor of CD4 + CD25 + Tregs.
To investigate the generation of Tregs induced by SJMHE1 on the immune system and inflammatory site further, we detected the expression of Foxp3 in the splenocytes and lungs of allergic mice.
As shown in Figure 5A

| D ISCUSS I ON
Parasite-derived molecules regulate the host immune responses with various mechanisms to ensure their survival while inhibiting excessive inflammatory responses, such as suppressing the development of allergic diseases, in their host. Parasite-derived products may be more effective and safer to humans than other chemical drugs because parasites can parasitize their host for a long time by regulating the immune response of the host. 22 To date, many commercial companies and private entities have produced and marketed helminths to treat inflammatory diseases. 23 In the present study, we demonstrated that S japonicum peptide SJMHE1 can significantly suppress OVA-induced airway inflammation and reduce inflammatory cells, including eosinophil infiltration F I G U R E 3 SJMHE1 treatment modulates the expression of cytokines from lungs of allergic mice. On day 35, the mice were killed, and the lungs from each mouse were tested for mRNA expression of IFN-γ (A), IL-4 (B), IL-5 (C), IL-17 (D), IL-10 (E), TGF-β (F) and IL-35 (G) by qRT-PCR. Results are presented as mean ± SEM of 12 mice from two independent experiments performed in triplicate wells. * P < .05, ** P < .01, *** P < .001 F I G U R E 4 SJMHE1 treatment modulates Th1/Th2/Th17/Treg response in splenocytes of allergic mice. On day 35, the mice were killed, and the splenocytes from each mouse were tested for Th1/Th2/Th17/Treg subsets by flow cytometry. A, CD4 + IFN-γ + Th1 cells, B, CD4 + IL4 + Th2 cells, C, CD4 + IL17 + Th17 cells and D, CD4 + CD25 + Foxp3 + Tregs in each group are shown. Data are representative of the experiments. E, The percentage of CD4 + IFN-γ + Th1 cells, F, the percentage of CD4 + IL4 + Th2 cells, G, the percentage of CD4 + IL17 + Th17 cells and H, the percentage of CD4 + CD25 + Foxp3 + Tregs in each group are shown. Results are presented as mean ± SEM of 12 mice from two independent experiments. * P < .05, ** P < .01, *** P < .001 into the airways. However, the reduction of airway inflammation induced by SJMHE1 is not associated with reduction in IgE levels because SJMHE1 treatment did not alter IgE levels in the OVA, OVA/ PBS and OVA/SJMHE1 groups. Helminth infection is known to induce both polyclonal-and antigen-specific IgE production and elicits an IgE-associated type 2 immune response. 24 As a S japonicum-derived peptide, SJMHE1 may induce IgE response. These results are consistent with other parasite studies demonstrating that excretory/ secretory (ES) and somatic products of Marshallagia marshalli reduce inflammatory cell infiltration and suppress OVA-induced allergic asthma but fail to decrease IgE levels. 25 Similarly, Kitagaki and colleagues reported that Heligosomoides polygyrus infection protects mice against asthma but increases OVA-specific IgE. 26 [34][35][36] Furthermore, it induces the production of IL-10 and IFN-γ. 37 In addition to IL-35, an increase in IFN-γ and IL-10 has been observed in Trichuris muris-infected mice, and this increase can inhibit the allergic airway inflammation induced by papain. 38 Although we did not investigate how IFN-γ, IL-10 and IL-35 affect and produce one another by SJMHE1 treatment, we found that the inhibition of IL-4, IL-5 and IL-17 and elevation of IFN-γ, IL-10 and IL-35 may contribute to the protection conferred by SJMHE1 in allergic mice. This study, which up-regulated the production of IL-10 and IL-35, is different from those on suppression induced by SJMHE1 in DTH and CIA mice. 16,17 We support the notion that helminths or their products regulate Th1/Th2/ Th17-associated inflammation in different models by modulating distinct targets. 39 It should be noted that cytokines are produced in splenocytes primarily by CD4 T cells because these cells affect inflammatory responses in asthma. 2 In line with the results in Figure 4, IFN-γ decreased, but IL-4 and IL-17A increased in OVA and OVA/PBS group than in PBS group gating from CD4 T cells as observed by flow cytometry. SJMHE1 treatment increased IFN-γ but decreased IL-4 from CD4 T cells. However, cytokine production in lungs from allergic mice is complex; apart from traditional Th2 cells, the lungs also contain epithelium-activated group 2 innate lymphoid cells (ILC2s). 40 To clarify the pathogenesis of asthma, the cell source of cytokines in splenocytes and lungs of allergic mice should be determined in the future. Listeria monocytogenes. 48 SJMHE1 appears to reset the balance of F I G U R E 6 SJMHE1 treatment up-regulates the expression of T-bet and down-regulates the expression of GATA3 and RORγt in lungs of allergic mice. On day 35, the mice were killed, and the lungs from each mouse were tested for T-bet, GATA3 and RORγt expression by Western blotting analysis. A representative blot from each group is shown effector T cells in inflammatory sites by acting upstream of inflammatory cascades. Thus, the inhibition of inflammation using helminths or their products is a consequence of the natural immune response to helminths. This inflammation inhibition induced by helminths does not target a single molecule or pathway; the suppression is the outcome of the complex and multifaceted immune response, the mechanism of which requires further understanding. We support the notion that helminths or their products can interact with a variety of innate and adaptive immune cells to disrupt pathogenic networks elicited by stromal cells in microenvironmental niches. 39 In addition to such therapeutic potential, these helminth-derived molecules can be used to validate the key regulators of disease pathogenesis. 49 Thus, small molecule peptides such as SJMHE1 from helminths are substantially more acceptable and easily regulated as a therapeutic modality than experimental helminth infections or their intact proteins. This class of biologics has appeal for a variety of inflammatory diseases, such as allergy and autoimmune diseases, which are usually elicited by an imbalance between pro-and anti-inflammatory T cell responses and are pandemics in industrialized and developing nations.
In summary, we demonstrated that SJMHE, a peptide from S japonicum, can inhibit airway inflammation in OVA-induced experimental asthma in mice. The up-regulation of Tregs and the Th1skewed responses induced by SJMHE1 in the immune system and inflammatory sites might provide protection against airway inflammation in allergic mice.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests. The funding agencies played no role in the design or implementation of the study, analysis or interpretation of the data, or the preparation and submission of the manuscript.