Allergen-Specific Treg Cells Upregulated by Lung-Stage S. japonicum Infection Alleviates Allergic Airway Inflammation

Schistosoma japonicum infection showed protective effects against allergic airway inflammation (AAI). However, controversial findings exist especially regarding the timing of the helminth infection and the underlying mechanisms. Most previous studies focused on understanding the preventive effect of S. japonicum infection on asthma (infection before allergen sensitization), whereas the protective effects of S. japonicum infection (allergen sensitization before infection) on asthma were rarely investigated. In this study, we investigated the protective effects of S. japonicum infection on AAI using a mouse model of OVA-induced asthma. To explore how the timing of S. japonicum infection influences its protective effect, the mice were percutaneously infected with cercaria of S. japonicum at either 1 day (infection at lung-stage during AAI) or 14 days before ovalbumin (OVA) challenge (infection at post–lung-stage during AAI). We found that lung-stage S. japonicum infection significantly ameliorated OVA-induced AAI, whereas post–lung-stage infection did not. Mechanistically, lung-stage S. japonicum infection significantly upregulated the frequency of regulatory T cells (Treg cells), especially OVA-specific Treg cells, in lung tissue, which negatively correlated with the level of OVA-specific immunoglobulin E (IgE). Depletion of Treg cells in vivo partially counteracted the protective effect of lung-stage S. japonicum infection on asthma. Furthermore, transcriptomic analysis of lung tissue showed that lung-stage S. japonicum infection during AAI shaped the microenvironment to favor Treg induction. In conclusion, our data showed that lung-stage S. japonicum infection could relieve OVA-induced asthma in a mouse model. The protective effect was mediated by the upregulated OVA-specific Treg cells, which suppressed IgE production. Our results may facilitate the discovery of a novel therapy for AAI.

Schistosoma japonicum infection showed protective effects against allergic airway inflammation (AAI). However, controversial findings exist especially regarding the timing of the helminth infection and the underlying mechanisms. Most previous studies focused on understanding the preventive effect of S. japonicum infection on asthma (infection before allergen sensitization), whereas the protective effects of S. japonicum infection (allergen sensitization before infection) on asthma were rarely investigated. In this study, we investigated the protective effects of S. japonicum infection on AAI using a mouse model of OVA-induced asthma. To explore how the timing of S. japonicum infection influences its protective effect, the mice were percutaneously infected with cercaria of S. japonicum at either 1 day (infection at lung-stage during AAI) or 14 days before ovalbumin (OVA) challenge (infection at post-lung-stage during AAI). We found that lung-stage S. japonicum infection significantly ameliorated OVA-induced AAI, whereas post-lung-stage infection did not. Mechanistically, lung-stage S. japonicum infection significantly upregulated the frequency of regulatory T cells (Treg cells), especially OVA-specific Treg cells, in lung tissue, which negatively correlated with the level of OVAspecific immunoglobulin E (IgE). Depletion of Treg cells in vivo partially counteracted the protective effect of lung-stage S. japonicum infection on asthma. Furthermore, transcriptomic analysis of lung tissue showed that lung-stage S. japonicum infection during AAI shaped the microenvironment to favor Treg induction. In conclusion, our data showed that lung-stage S. japonicum infection could relieve OVA-induced asthma in a mouse model. The protective effect was mediated by the upregulated OVA-specific Treg cells, which suppressed IgE production. Our results may facilitate the discovery of a novel therapy for AAI.

INTRODUCTION
The prevalence of asthma has increased dramatically in the past three decades (Eder et al., 2006;Ali, 2011) and represents a great health burden, especially in developed countries (Barnes, 2004;Thomsen, 2015). Atopic asthma is the most common form of asthma. It is an immunological disorder characterized by inflammation of the airways and lungs triggered by allergen with marked T H 2 responses, overactive immunoglobulin E (IgE) production, mucus hypersecretion, and large amount of eosinophil influx to the airways (Lambrecht and Hammad, 2015).
The exact social and environmental factors that lead to the hyperreactive immune disorder is not fully understood. A leading theory behind the rapid rise of allergy and asthma rates is the "hygiene hypothesis, " which suggests that the decreasing incidence of infections in Western countries may contribute to the rise of both autoimmune and allergic diseases (Okada et al., 2010). This hypothesis was supported by an observation showing that the Western lifestyle was linked with significantly higher prevalence of atopic diseases (Herbert et al., 2009). A putative explanation to this phenomenon is that the overall reduction in common T H 1-inducing (bacterial, viral, and parasitical) infections results in a decreased ability to counterbalance T H 2polarized allergic diseases (Umetsu, 2012;Yang et al., 2016;Stiemsma and Turvey, 2017). Following this lead, a variety of experimental studies have shown that helminth infections can downregulate host immunity and immunopathology in allergy and other immune disorders (Sitcharungsi and Sirivichayakul, 2013;Maizels, 2016;Maizels and McSorley, 2016). Schistosome was one of the parasites that have been found to have protective effects against autoimmune diseases and allergies such as arthritis and asthma (Osada et al., 2009;Janssen et al., 2016;Qiu et al., 2017). These explorations hold great promise in identifying a new and more specific intervention measure for atopic asthma that does not result in certain side effects, such as increased susceptibility to infection and necrosis, which can be triggered by steroid hormone drugs such as dexamethasone (Kuprys-Lipinska and Kuna, 2014;Al-Ahmad et al., 2018;Falk, 2018).
Schistosome is an ancient parasite affecting more than 230 million people in 78 tropical and subtropical countries (LoVerde, 2019). During its life stages in definitive hosts, schistosome invades its mammalian hosts through the skin, migrates from skin to lung, and then develops and matures in the liver, finally residing in the mesenteric venules (McManus et al., 2018). Although it has been shown by multiple studies that schistosome could abate allergic airway inflammation (AAI), the understanding of its underlying mechanisms remains limited. Most previous studies focused on testing the preventive effect (infection before allergen sensitization) of the Schistosoma japonicum infection against allergic asthma. Under this setting, controversial results have been reported regarding both the timing of the infection (acute vs. chronic) (Smits et al., 2007;van der Vlugt et al., 2012;Layland et al., 2013) and the effector component (egg vs. worms) (Mangan et al., 2006;Pacifico et al., 2009;Obieglo et al., 2018), which reflects the complexities of the schistosome life cycle and its immune regulatory components. Moreover, contradictory results were also reported regarding the roles of regulatory T cells (Treg cells) in schistosomemediated protection. Some studies showed that Treg cell was an important effector in schistosome-mediated protection against asthma (Medeiros et al., 2003;Smits et al., 2007;Pacifico et al., 2009;Layland et al., 2013;Zhang et al., 2019), whereas a more recent study showed that the protection was independent of Treg cells (Obieglo et al., 2018).
Unlike previous studies that were focused on testing the preventive effect (infection before allergen sensitization) of S. japonicum infection against allergic asthma, the primary goal of this study was to investigate the protective effect of S. japonicum infection on asthmatic inflammation (infection after allergen sensitization) and to clarify the underlying mechanism. To this aim, mice were percutaneously infected with cercaria of S. japonicum at either 1 day before OVA-induced asthma attack (infection at lung-stage during AAI) or 14 days before OVA-induced asthma attack (infection at post-lung-stage during AAI). We found that only lung-stage S. japonicum infection could upregulate the frequency of allergen-specific Treg cell, which significantly alleviated AAI by inhibiting IgE production and inflammatory cytokine secretion.

Ethics Statement
All experiments and methods were performed in accordance with relevant guidelines and regulations. Mice experiments were carried out at the National Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention (NIPD, China CDC) in Shanghai, China. All animal experiment protocols used in this study were approved by the Laboratory Animal Welfare & Ethic Committee of the National Institute of Parasitic Diseases (permit no. IPD-2016-7).

OVA-Induced AAI and S. japonicum Infection
Female BALB/c mice (6-8 weeks old) were randomly divided into six groups in this experiment, which were OVA-induced AAI (OVA) group, OVA-induced AAI with lung-stage S. japonicum infection (OVA + INF, lung stage) group, OVA-induced AAI with post-lung-stage infection (OVA + INF, post-lung stage) group, and OVA-induced AAI with dexamethasone (DXM) treatment (OVA + DXM) group, as well as infection (INF) group and normal (NOR) group. For OVA-induced AAI, the mice were sensitized by injecting 10 µg of alum-adjuvanted ovalbumin (OVA; cat. # 77120 and 77161; Thermo Fisher, United States) intraperitoneally on days 0 and 14. Subsequently, the mice were challenged with aerosolized OVA [1% in phosphate-buffered solution (PBS)] for 30 min in the chamber of a Medical Compressor Nebulizer (DEDAKJ, Germany) on days 21 to 24 (Figures 1A,B). The mice of the normal control and S. japonicum infection control groups were challenged with PBS. To test the protective effect of infection on OVA-induced AAI, mice were infected with 15 cercaria of S. japonicum at either 1 day (infection within 6 days: lung-stage infection) or 14 days before Inflammatory cell (total cell, macrophage, eosnophils, lymphocytes, neutrophils) infiltration in BALF of mice after OVA challenge was compared in NOR, INF, OVA, OVA + INF, and OVA + DXM groups (n = 5 or 6 mice per group, experiment performed twice). (E,F) Representative images of H&E and PAS staining of lung tissue after OVA challenge. Statistical analysis of inflammation score and mucus secretion score are also shown in (E,F), respectively, among five groups (n = 5 or 6 mice per group, experiment performed twice). NOR, normal mice (without OVA sensitization and challenge); INF, mice without OVA sensitization and challenge but infected with schistosome; OVA, mice with OVA sensitization and challenge but without S. japonicum infection; OVA + INF, mice sensitized and challenged with OVA and treated with S. japonicum infection; OVA + DXM, mice sensitized and challenged with OVA and treated with dexamethasone. All data are shown as mean ± SEM. *P < 0.05, **P < 0.01; NS, not significant by one-way analysis of variance (ANOVA) with Tukey test. #, ##, and ### indicate P < 0.05, <0.01, and <0.001, respectively, OVA versus NOR (C,D). *, **, *** indicated P < 0.05, <0.01, and <0.001, respectively, OVA + INF or OVA + DXM versus OVA (C,D).

Bronchoalveolar Lavage Collection and Cell Counting
Mice were euthanized 48 h after the last aerosolized OVA challenge (day 26), and bronchoalveolar lavage fluids (BALFs) were collected as previously reported method (Li et al., 2012). Briefly, after euthanasia, tracheotomy was carried out, and an arteriovenous indwelling needle (20-gauge; BRAUN, Germany) was inserted into the trachea. Lavages were collected by washing the lung twice with 0.3 mL PBS. Cells in BALFs were harvested after centrifugation, and the supernatants were stored at -80 • C for cytokine detection. Cell pellet was fixed with paraformaldehyde (4%) and stained with a hematoxylin-eosin (H&E). A total of 1,000 cells from multiple fields were examined for each slide. Counts of total cells, eosinophils, macrophage, neutrophils, and lymphocytes were performed on blinded samples, as described previously (Chang and Yen, 2004).

Lung Histopathology
Lung tissues were fixed in 4% phosphate-buffered formaldehyde overnight and then embedded in paraffin and cut for H&E and periodic acid-Schiff (PAS) staining. Images of the stained sections were captured with a NIKON DS-U3 microscope (NIKON, Japan). Lung inflammation and the intensity of goblet cell metaplasia were assessed and scored 0 to 4 by two blinded, independent investigators, as described previously (Hopfenspirger and Agrawal, 2002).

Determination of Total and OVA-Specific IgE in Serum
The levels of total and OVA-specific IgE in serum were measured using enzyme-linked immunosorbent assay. Briefly, Maxisorp 96-well microtiter plates (Thermo Fisher Scientific, United States) were coated with rat monoclonal anti-mouse IgE antibody for total IgE detection (1:1,000; cat. # ab99571, Abcam, United Kingdom) or 10 µg/mL OVA for OVA-specific IgE (cat. # A5503, Sigma, United States) 100 µL/well, respectively, in carbonate-bicarbonate buffer, pH 9.6, for 12-16 h at 4 • C. Then, the plates were blocked for at least 2 h at 37 • C with 100 µL/well of PBS plus bovine serum albumin (BSA) (1%). After wash, 100 µL serum diluted with PBS containing 0.05% Tween 20 (PBST) (1:40 for total IgE; 1:5 for OVA-specific IgE) was added to each well and incubated at 37 • C for 2 h. Next, horseradish peroxidaselabeled goat anti-mouse IgE antibody was diluted with PBST (1:2,000; cat. # ab99574, Abcam, United Kingdom) and added to each well at 100 µL/well. After 2 h of incubation at 37 • C, the plates were washed with PBST five times. Finally, color was developed by addition of 100 µL/well of TMB (cat. # PA107, TIANGEN, China), and after incubation at room temperature for maximal 30 min, the reaction was stopped with 5% sulfuric acid (50 µL/well). Optical density values were determined at 450 nm using the multimode microplate readers (BioTek, United States). The concentration of total IgE was then calculated according to the standard curve.

Lymphocyte Isolation From Lung Tissues
After collection, lung tissues were washed three to four times with RPMI (Roswell Park Memorial Institute) medium, minced to tiny pieces, and then digested in 0.1% type IV collagenase (cat. # C8160, Solarbio, China) solution at 37 • C for 30 min. Digested lung tissues were filtered through a 70-µm cell strainer, and erythrocytes were lysed with a red blood cell lysis buffer (cat. # R1010, Solarbio, China).
Adoptive Transfer of Naive CD4 + T Cells Naive CD4 + T cells of CD45.1 + OT II mice were purified using EasySep Mouse Naive CD4 + T Cell Isolation Kit (cat. # 19765, StemCell, United States) according to the manufacturer's protocol. The purity of isolated cells was checked by flow cytometry and was confirmed to be >85%. Freshly purified naive CD4 + T cells were suspended in PBS and injected intravenously into CD45.2 + congenic C57BL/6 recipient mice, 1 × 10 6 cells/mouse. The induction of AAI and S. japonicum infection was performed as described above.
(A,C) OVA-specific IgE in sera were measured by enzyme-linked immunosorbent assay from NOR, INF, OVA, OVA + INF, and OVA + DXM groups (n = 5 or 6 mice per group, experiment performed twice). (B,D) The concentrations of total IgE in mouse serum were compared among all groups after OVA challenge (n = 5 or 6 mice per group, experiment performed twice). All data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant by one-way analysis of variance (ANOVA) with Tukey test.
were randomly divided into two groups: OVA + INF + αCD25 and OVA + INF + IgG. OVA sensitization, aerosol challenge, and S. japonicum infection were performed as described above.

RNA Sequencing
Total RNA was extracted from lung tissues by using Trizol reagent (cat. # 15596026, Invitrogen). RNA purity was checked using the Nano Photometer spectrophotometer (IMPLEN, CA, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States); 1 µg total RNA from each sample was used to construct the sequencing library using

Differential Expression Genes Analysis and Functional Enrichment Analysis
Sequencing quality was evaluated by FastQC software 1 . Poorquality reads and adaptors were trimmed by Trimmomatic software (released version 0.22 2 ), and only reads longer than 50 bp were used for further analysis. The high-quality reads were mapped to mouse genome (mouse BALB/cJ) downloaded in Ensembl database. The HTseq (Anders et al., 2015) was used to quantify gene expression, and R DEseq2 package (Anders and Huber, 2010) was employed for differential expression analysis. Only genes with false discovery rate (FDR) adjusted P < 0.05 and absolute value of fold change > 2 were considered as differential expression genes (DEGs). Functional enrichment of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs were conducted by R Cluster Profiler package (Yu et al., 2012) with FDR correction. Significantly enriched GO terms and KEGG pathways were identified with corrected P < 0.05. DEG-related pathways enrichment terms were performed with the Panther Classification System 3 . 2 http://www.usadellab.org/cms/index.php?page=trimmomatic 3 http://pantherdb.org/ Data and materials availability: RNA sequencing data are deposited in the SRA database SRA (SRA accession no. PRJNA609083).

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, United States). The data of quantitative variables were presented as mean ± standard error of mean (SEM). P < 0.05 was considered statistically significant.

RESULTS
Lung-Stage S. japonicum Infection Ameliorated OVA-Induced AAI in a Murine Model A mouse model of OVA-induced AAI was adopted to test the protective effect of S. japonicum infection on allergic asthma ( Figures 1A,B). Compared to the control group, mice in the OVA group showed significant infiltration of inflammatory cells in BALFs (Figures 1C,D), which resembled the main clinical feature of AAI (Persson, 2019). Moreover, after S. japonicum infection, the results showed that the lung-stage infection significantly reduced the infiltration of inflammatory cells, especially eosinophils (Figure 1C), whereas post-lung-stage infection did not ( Figure 1D). Histopathologic examination further confirmed the above findings by showing that lungstage infection significantly suppressed the OVA-induced eosinophil-rich leukocyte infiltration and mucus hypersecretion (Figure 1E), whereas post-lung-stage infection showed no obvious protective effect ( Figure 1F).

Lung-Stage S. japonicum Infection Inhibited IgE Production and Suppressed T H 2 Cytokine Secretions
IgE is the key factor mediating the pathological immune responses that lead to allergic asthma (Galli and Tsai, 2012). To further characterize the protective effects of S. japonicum infection, we measured the total and OVA-specific IgE in serum of mice. The results showed that lung-stage infection significantly downregulated both the total and OVA-specific IgE to levels comparable with DXM treated mice (Figures 2A,B). In contrast, post-lung-stage infection tended to elevate the total and OVA-specific IgE levels despite no significant difference was reached (Figures 2C,D). Moreover, we also measured a panel of cytokines and chemokines in BALFs and found that lung-stage infection altered the cytokine/chemokine secretion pattern induced by aerosolized OVA challenge ( Figure 3A and Supplementary Figure 1). More specifically, IL-5 and eotaxin were reduced to levels that are similarly attained with DXM treatment (Figure 3B). On the contrary, post-lung-stage infection increased IL-4 and IL-5 secretion ( Figure 3B).

Lung-Stage S. japonicum Infection Upregulated the Frequencies of Treg Cells Especially OVA-Specific Treg Cells in Lung
Treg cell was suggested to be a key factor of S. mansoni-mediated protection against AAI (Layland et al., 2013). Here, we first assessed the frequencies of total Treg cells (CD4 + CD25 + Foxp3 + Treg cell) in spleen and lung. As shown in Figure 4A, lung-stage infection upregulated the frequency of total Treg cells both in lung and spleen (Figure 4A), whereas post-lung-stage infection only slightly improved the proportion of Treg cells in the spleen ( Figure 4B). Then, by adoptive transfer of OVA-specific naive CD4 + T cells (CD45.1 + ) into wide-type CD45.2 + mice ( Figure 5A), we found that the frequency of OVA-specific Treg cells (CD45.1 + Treg) in lung increased by more than threefold  after S. japonicum infection (P < 0.001), whereas the frequency of endogenous Treg cells (CD45.2 + Treg cell) in lung was not significantly improved (Figures 5B,C). The proportion of total Treg cells was increased in lung and LDLNs after S. japonicum infection (Figures 5B,C).
We also found that the ratio of OVA-specific IL-4 + versus IFN-γ + CD4 + T cells significantly decreased after lung-stage S. japonicum infection (Supplementary Figure 2), suggesting that specific CD4 + T cell responses shifted from T H 2 toward T H 1 responses.

The Protective Effect of Lung-Stage S. japonicum Infection Was Treg Cell-Dependent
Significant negative correlations between the frequency of Treg cells and OVA-specific IgE or IgG (Figure 6) were observed, indicating that the protective effect of S. japonicum infection on AAI might be mediated by Treg cell. To elucidate the role of Treg cell, we performed in vivo depletion of Treg cells using antimouse CD25 antibody ( Figure 7A). The efficiency of Treg cell deletion is demonstrated by the significantly reduced percentage of CD25 + Treg cells as shown in Supplementary Figure 3. Our data showed that Treg depletion (OVA + INF + αCD25 group) aggravated OVA-induced AAI compared to isotype control group. Inflammatory cell infiltration, mucus secretion (shown by PAS staining), OVA-specific IgE production, and eotaxin secretion significantly increased after Treg depletion (Figure 7).

Lung-Stage S. japonicum Infection Molded the Microenvironment to Facilitate the Generation of Treg Cells
To reveal factors that contributed to the induction of Treg cells upon lung-stage S. japonicum infection, we created transcriptomic profiles of the lung tissues from the schistosome infected and non-infected mice after OVA challenge. The results showed that 203 genes were upregulated, and 279 genes were downregulated after lung-stage S. japonicum infection ( Figure 8A and Supplementary Data File 1). GO analysis of DEGs showed that the top three terms of significantly enriched genes (P < 0.05) are mainly distributed in the T cell activation, the leukocyte proliferation, and the regulation of leukocyte proliferation (Figure 8B) pathways. Panther analysis showed that 84 DEGs are related to immune system processes (Supplementary Figure 4), and 70 of them were downregulated (Supplementary Data File 1). Further analysis showed that three genes (CD46, Epor, and Klra17) reported to promote Treg cell response were upregulated (Gehrie et al., 2011;Tsai et al., 2012;Purroy et al., 2017), and eight genes (Clec7a, CCR6, Spi-B, ABCG1, ADA, Ctsk, Ctss, and Ptgir) reported to inhibit Treg cell response were downregulated (Liu et al., 2013;Tang et al., 2015;Cheng et al., 2016;Naval-Macabuhay et al., 2016;Rauch et al., 2016;Yan et al., 2017;Zhou et al., 2017;Kulkarni et al., 2018; Figure 8C and Table 1) in schistosome-infected mice. We postulated that lung-stage S. japonicum infection generated a microenvironment facilitating Treg cell development in lung ( Figure 8C).

DISCUSSION
The eradication of helminths (and other pathogens) is suggested to have resulted in decreased immune-regulatory ability, which might be the cause of the increasing prevalence of allergic and autoimmune disorders especially in developed and urbanized countries (de Ruiter et al., 2017;Harnett and Harnett, 2017;  Comparisons of total inflammatory cell counts in BALF between lung-stage schistosome-infected mice treated with either anti-CD25 antibody or isotype control IgG (n = 8 mice per group, experiment performed twice). (C) Lung histopathology analysis of lung-stage schistosome-infected mice treated with either anti-CD25 antibody or isotype control IgG. Upper, H&E staining; lower, PAS staining (n = 8 mice per group, experiment performed twice). (D) Comparisons of OVA-specific IgE and IgG in sera between Treg cell-depleted and control mice (n = 8 mice per group, experiment performed twice). (E) Comparisons of eotaxin levels in BALF between lung stage-infected mice treated with either anti-CD25 antibody or isotype control (n = 8 mice per group, experiment performed twice). Data are shown as mean ± SEM. *P < 0.05, ***P < 0.001 by one-way analysis of variance (ANOVA) with Tukey test. Bach, 2018). The protective effect of parasitic infection against allergies and autoimmune disease has been extensively explored especially after the hygiene hypothesis was introduced into this field (Maizels et al., 2014), among which the immunoregulation of schistosome is best illustrated (Capron, 2011;Layland et al., 2013;Qiu et al., 2017). In this study, to investigate how the timing of S. japonicum infection influenced the development of allergic asthma, we compared the protective effect of two phases of S. japonicum infection: lung stage and post-lung stage. We found that lung-stage S. japonicum infection significantly relieved OVAinduced AAI, but post-lung-stage infection showed no protective effect. Within lung-stage infection (3-7 days postinfection), schistosomula transformed from cercaria were completely located in lung tissue of the host (Rheinberg et al., 1998), which might modulate the local immune response to abate OVAinduced AAI. We postulated that this might be the reason that the protective effect of lung-stage infection was superior to post-lung-stage infection. And indeed, we found that lungstage infection significantly upregulated Treg cell response in lung tissues.
Multiple factors such as worm species, timing, intensity and chronicity of infection, and host genetics have been investigated to illustrate the mechanisms of helminth-mediated regulation of host immunity (Cooper, 2009). Nonetheless, the relationship between helminths and asthma still remains not well understood.
Mechanistic studies reported contradictory results; for example, one study showed that S. mansoni-mediated suppression of AAI was patency dependent and mediated by infection-induced Treg cells (Layland et al., 2013), whereas another study showed that protection mediated by S. mansoni egg was independent of either Treg cells or Breg cells (Obieglo et al., 2018). In the current study, we found that lung-stage S. japonicum infection that occurred during OVA challenge could upregulate the frequency of Treg cells and suppress OVA-specific IL-4 response. Upregulation of Treg cells by S. japonicum infection has been reported by few previous studies (Baru et al., 2010;Layland et al., 2013); however, to our knowledge, this is the first proof showing that the lung-stage S. japonicum infection can upregulate allergen OVA-specific Treg cell.
To elucidate the role of Treg cells in S. japonicum infectionmediated alleviation of AAI, we first analyzed the relationship between Treg cells and OVA-specific IgE and found that the frequency of Treg cells in lung negatively correlated with OVAspecific IgE. The suppression of IgE secretion by Treg cells has been observed and described by multiple previous studies  (Meiler et al., 2008;Wing et al., 2008;Khan, 2020). IL-10 and CTLA-4 pathways were suggested to be associated with the suppression of IgE; however, the detailed molecular mechanism is still elusive. Furthermore, by in vivo depletion of Treg cell, we found that the decrease in IgE secretion was Treg celldependent. IgE acts as the major mediator contributing to AAI (Gabet et al., 2019). Our results demonstrated that the protective effect of S. japonicum infection on AAI was mediated by Treg cell-dependent inhibition of IgE, which was consistent with a previous report showing that the preventive effect of chronic S. mansoni infection against later AAI was also Treg cells dependent (Layland et al., 2013). Mechanisms underlying the induction of Treg or Breg cells by helminth-related antigens have been reported (Zaccone et al., 2009;Haeberlein et al., 2017). However, we did not find out the exact active molecules of schistosome that led to the upregulation of Treg cells in this study. Nonetheless, we think that it is very likely that the observed protective effect was a collective result of multiple components of the schistosome, as previous studies showed that multiple enzymes released by schistosomula could regulate host immunity (Hansell et al., 2008;Liu et al., 2015). We plan to acutely define these components in the future.
Instead of identifying effector antigens, in this study, we tried to understand how the lung-stage S. japonicum infection influences local immune responses in lung. To do so, we performed a transcriptomic comparison between lung tissues of schistosome-infected and non-infected mice. The results showed that, after lung-stage S. japonicum infection, most genes related to immune response were downregulated (70/84), which implied that the general immune state in lung tended to be downregulated by S. japonicum infection. Among these genes, we found that three genes (CD46, Epor, and Klra17) reported to promote Treg cell response were upregulated, and eight genes (Clec7a, CCR6, Spi-B, ABCG1, ADA, Ctsk, Ctss, and Ptgir) reported to inhibit Treg cell response were downregulated in schistosome-infected mice, suggesting that S. japonicum infection generated a milieu facilitating Treg cell induction in the lung. In the meantime, we also observed that some molecules reported to facilitate the function of B cells or plasma cells were downregulated, which was consistent with our finding that IgE response was suppressed.
Collectively, our study showed that lung-stage S. japonicum infection established a regulatory environment in the lungs, which can help to relieve OVA-induced AAI in a mouse model. Although the exact mechanism of Treg cell upregulation remains elusive, our data showed that lung-stage S. japonicum infection can improve the population of allergen-specific Treg cells that suppress IgE production. These results highlight the value of lung-stage S. japonicum infection as a potential therapy for allergic asthma.

DATA AVAILABILITY STATEMENT
RNA sequencing data are deposited in the SRA database SRA, SRA accession number: PRJNA609083.

ETHICS STATEMENT
The animal study was reviewed and approved by Laboratory Animal Welfare & Ethic Committee (LAWEC) of National Institute of Parasitic Diseases (Permit Number: IPD-2016-7).

AUTHOR CONTRIBUTIONS
ZL, WZ, WY, BZ, YX, XW, YW, QW, CS, and JL conducted the experiment. ZL, YW, and WH designed the experiment and analyzed the data. FL analyzed the data of RNA-sequence. ZW, FQ, JC, and BX provided intellectual input and aided the experimental design. ZL wrote the manuscript. YW and WH revised the manuscript. All the authors contributed to the article and approved the submitted version.