Suppression of airway allergic eosinophilia by Hp‐TGM, a helminth mimic of TGF‐β

ABSTRACT Type 2‐high asthma is a chronic inflammatory disease of the airways which is increasingly prevalent in countries where helminth parasite infections are rare, and characterized by T helper 2 (Th2)‐dependent accumulation of eosinophils in the lungs. Regulatory cytokines such as TGF‐β can restrain inflammatory reactions, dampen allergic Th2 responses, and control eosinophil activation. The murine helminth parasite Heligmosomoides polygyrus releases a TGF‐β mimic (Hp‐TGM) that replicates the biological and functional properties of TGF‐β despite bearing no structural similarity to the mammalian protein. Here, we investigated if Hp‐TGM could alleviate allergic airway inflammation in mice exposed to Alternaria alternata allergen, house dust mite (HDM) extract or alum‐adjuvanted ovalbumin protein (OVA). Intranasal administration of Hp‐TGM during Alternaria exposure sharply reduced airway and lung tissue eosinophilia along with bronchoalveolar lavage fluid IL‐5 and lung IL‐33 cytokine levels at 24 h. The protective effect of Hp‐TGM on airway eosinophilia was also obtained in the longer T‐cell mediated models of HDM or OVA sensitisation with significant inhibition of eotaxin‐1, IL‐4 and IL‐13 responses depending on the model and time‐point. Hp‐TGM was also protective when administered parenterally either when given at the time of allergic sensitisation or during airway allergen challenge. This project has taken the first steps in identifying the role of Hp‐TGM in allergic asthma and highlighted its ability to control lung inflammation and allergic pathology. Future research will investigate the mode of action of Hp‐TGM against airway allergic eosinophilia, and further explore its potential to be developed as a biotherapeutic in allergic asthma.


INTRODUCTION
Type 2-high asthma is a chronic inflammatory disease of the airways leading to cough, wheeze, shortness of breath, and chest tightness. This form of asthma is induced by early-life encounters with environmental allergens, including house dust mite (HDM) and Alternaria alternata, which evoke T helper 2 (Th2) cell responses [1]. Exuberant production of IL-4, IL-5, and IL-13 leads to asthma features with the accumulation of type 2-associated cells, such as eosinophils, in lung tissue [2,[3][4][5]. Eosinophil infiltration into the lung plays a causal role in augmentation of broader inflammation and is associated with asthma disease severity [1,6,7]. Targeting eosinophils has shown promise in alleviating asthma in clinical trials with therapeutic antibodies blocking type 2 cytokines or their receptors [3,4,[8][9][10][11][12][13][14].
Eosinophils are regulated by a suite of cells, cytokines, and chemokines. In response to inhaled allergens, alarmin cytokines such as IL-33 are released by necrotic airway epithelial cell, triggering type 2 dendritic cells, which migrate from the lung tissue to the draining lymph node to induce Th2 differentiation [15]. Type 2 innate lymphoid cells (ILC2s) also respond to IL-33, and together with Th2 cells produce interleukin (IL)-5, to induce eosinophil differentiation, activation and recruitment into inflamed lungs [16]. Contemporaneously, IL-4 and IL-13 production stimulates the generation of eotaxins (e.g., Eotaxin-1/CCL-11), the main chemoattractants for eosinophils produced by lung epithelial cells, fibroblasts and smooth muscle cells [4,17].
IL-5 is considered to be the major cytokine in eosinophil biology, mobilizing eosinophils from the bone marrow during allergic inflammation, and driving their activation and proliferation in peripheral tissues [1,13,18]. Together with IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-5 can prolong eosinophil survival and abrogate their apoptosis [19]. In addition, specific subsets of dendritic cells (type 1 dendritic cells) attract eosinophils into the airways in asthma models, mediated by the chemokines CCL17 and CCL22 [20].
Type 2-high allergic asthma is increasingly prevalent in the more economically developed countries, particularly in urban communities, where helminth parasite infections are rare. Helminth parasites may broadly suppress type 2 responses to escape the arm of immunity that targets them for expulsion. Interestingly, children with helminth infections show reduced levels of allergic reactivity [40], which is linked to the expansion of Tregs [41] and their ability to suppress airway allergy in mouse models [36,42]. Furthermore, treatment with anthelmintic drugs to clear parasites results in more rapid acquisition of allergic reactivity [43,44]. We and others have screened helminth parasites for molecular mediators that might explain reduced allergic status; such products are likely to have been honed by evolution into effective biological inhibitors of the type 2 immune pathway that may offer novel treatments for human asthma [45,46].
In this study, we investigated the effects of Hp-TGM in murine models of allergic asthma elicited by A. alternata allergen, HDM or alum-adjuvanted Ovalbumin (OVA) protein. In the simplest model, intranasal administration of Hp-TGM in acute pulmonary inflammation following A. alternata exposure reduced pulmonary eosinophilia 24 h later. Moreover, in models of allergen challenge of sensitized mice, systemic administration of Hp-TGM potently reduced eosinophilic influx in the broncho-alveolar lavage fluid (BALF) and lung, both when given at the time of airway sensitization or at the time of airway challenge. Our data open the door to future research into therapeutic application of helminthderived molecules to prevent the development or alleviate the symptoms of allergic asthma.

Reagents
Alternaria alternata extract (Greer Laboratories XPM1D3A25) was resuspended in PBS and concentration assessed by Pierce BCA assay (Thermo Fisher Scientific). HDM extract from Dermatophagoides pteronyssinus (Greer Laboratories XPB70D3A2.5) was resuspended in sterile PBS. Class IV Ovalbumin (Sigma, Gillingham, Dorset, UK) was lipopolysaccharide (LPS) depleted by Triton X-114 phase separation, and the levels of LPS present in HES and depleted OVA were below 0.1 and 0.01 IU LPS per mg protein, respectively by the Limulus Amoebocyte Lysate assay (Lonza, Slough, UK).

Protein expression and purification
Construct encoding Hp-TGM was cloned into the pSec-TAG2A expression vector as previously described, with C-terminal myc and 6-His tags [53]. Purified plasmids were transfected into Expi293F™ cells, and supernatants collected 5 days later. Expi293F™ cells were maintained, and transfections carried out using the Expi293 Expression System according to manufacturer's instructions (ThermoFisher Scientific). Expressed protein in supernatants were purified over a HisTrap excel column (GE Healthcare) and eluted in a gradient up to 500 mM imidazole. Eluted protein was then dialysed to PBS and filter-sterilized. The protein concentration was determined from A280 nM (Nanodrop, ThermoFisher Scientific), using a calculated extinction coefficient.

Animals
BALB/cAnNCrl mice were purchased from Charles River, UK. RAG1 À/À mice and Foxp3-GFP mice were bred at the University of Glasgow. All mice were accommodated and procedures approved by the University of Glasgow Animal Welfare and Ethical Review Board, and performed under UK Home Office licences with institutional oversight performed by qualified veterinarians.

Murine lung and BALF single cell suspension preparation
Lungs were flushed with four washes of 0.5 ml ice-cold PBS to collect BALF cells, followed by lung dissection for single cell preparation. Murine lungs were digested in 2 U/mL of Liberase TL (Roche, Burgess Hill, UK) and 80 U/mL DNase (Life technologies, Paisley, UK) at 37 C with agitation for 35 min. Digested tissue was passed through a 70 μm strainer and red blood cells lysed using Red Blood Cells Lysis Buffer (Sigma). Live cells were counted using a haemocytometer and dead cells excluded using trypan blue.

Alternaria model
Alternaria allergen was used as a model of asthma as previously described [51,57]. Alternaria allergen (10 μg) and Hp-TGM (5 μg) were administered intranasally to mice under isoflurane anaesthesia and tissues were harvested 24 h after initial Alternaria allergen administration. House dust mite model HDM allergen was used as a model of asthma as previously described [58]. Mice were anaesthetized with isoflurane, sensitized intranasally (i.n.) with 3 μg HDM in 50 μl PBS. After 7 days, mice were challenged with 10 μg HDM on five consecutive days under anaesthesia. Three days after the last challenge, mice were sacrificed, and tissues were harvested. Hp-TGM (1 μg) was administered i.n. or intraperitoneally (i.p.), as indicated.

RNA extraction, reverse transcription and qPCR
Lung tissue was placed in RNALater Stabilizing Solution (Thermo Fisher Scientific) and stored at À20 C. Lung tissues were then transferred from RNALater to TRIzol (Thermo Fisher Scientific) and homogenized using 3 mm stainless steel beads (Qiagen) in a TissueLyser II (Qiagen) at 25 Hz for 2 min.
Complementary DNA was made using qScript ® cDNA Synthesis Kit (Quantabio). Primers were purchased from Life Technologies and amplification reaction was carried out using PerfeCTa ® SYBR ® Green Super-Mix, Low ROX (Quantabio) in a 13 μl volume made up of 0.25 μl of nuclease free water, 0.25 μl forward primer, 0.25 μl reverse primer, 6.25 μl of 2Â concentrated reaction mix and 6 μl DNA template (10 ng). Amplification was carried out using QuantStudio and 384-well plate (Applied Biosystems). PCR data were analysed using QuantStudio Real-Time PCR software v1.1 and the 2-ΔΔCT method. In brief, relative gene expression between different samples was calculated using the threshold cycles (CTs) generated by QuantStudio 384-well plate machine and software. ΔCT for each sample was calculated subtracting CT of the housekeeping gene (HPRT-1) from the CT of the gene of interest. To obtain the relative gene expression between control group and treatment, ΔΔCTs were then obtained subtracting the average of the control group ΔCTs (e.g., PBS) from the ΔCT of the sample. Subsequently, 2-ΔΔCT was calculated and plotted in a graph.

Statistical analysis
All data were analysed using Prism (Graphpad Software Inc.), one-way ANOVA with Tukey's multiple comparisons post-test was used to compare multiple independent groups. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = not significant (p > 0.05). Where significant effects were observed for physiological outcomes (e.g., eosinophilia) similar group sizes were deemed sufficient to test individual parameters, such as cytokines.

Hp-TGM inhibits innate allergic airway responses
We first evaluated the ability of Hp-TGM to influence the early innate pro-allergic response to the fungal allergen A. alternata 24 h after intranasal administration. This model was previously shown to be highly sensitive to inhibition by total H. polygyrus HES, and key IL-33-pathway inhibitors in HES, HpARI and HpBARI [51,52,57]. In this system, we tested the effect of coadministration of Hp-TGM and A. alternata extract by the intranasal route ( Figure 1a). We analysed differential cell responses by flow cytometry using the gating strategies shown for BALF (Supplementary Figure 1) and lung (Supplementary Figure 2) cells. BALF contents broadly represent responses of airway epithelial cells and resident or recruited immune cells to allergen exposure, while analyses of whole lung homogenates also reflect changes in the lung parenchyma.
In the BALF, A. alternata induced strong airway inflammation and eosinophilia which was highly suppressed by co-administration of Hp-TGM, as measured by total cellular infiltrate into the BALF (Figure 1b) and total eosinophils within the infiltrating population ( Figure 1c,d). In the lung, eosinophilia developed within 24 h of A. alternata treatment, but this was abolished by co-administration of Hp-TGM (Figure 1e). Hp-TGM was also effective in abrogating A. alternata-induced eosinophilia in RAG-1-deficient mice (Figure 1f,g), confirming that innate responses were suppressed directly without involvement of adaptive lymphocytes. Furthermore, Hp-TGM inhibition of A. alternata-induced pulmonary eosinophilia was observed to act in a dose-dependent manner with diminution evident even when 5 ng of the parasite mediator was administered (Supplementary Figure 3A). We then measured type 2 cytokines in BALF and lung homogenate; in BALF, IL-5 levels induced by A. alternata were substantially reduced when the allergen was coadministered with Hp-TGM (Figure 2a). BALF IL-13 levels were marginally above the limit of detection in some, but not all animals (data not shown), and Hp-TGM exerted no significant effect. In the lung, IL-33 in lung homogenates was fully suppressed to baseline levels in the presence of Hp-TGM (Figure 2b), while IL-5, IL-13 and eotaxin-1 were reduced to different degrees, albeit falling short of significance (Figure 2c-e). We also analysed type 2 innate lymphoid cells (ILC2s), in lung homogenates determined by flow cytometry. (f and g) Numbers of eosinophils in BALF (f) and lung homogenates (g) of RAG-1 deficient mice determined by flow cytometry. Results are pooled from two independent experiments each with 2-4 mice per group. All data were analysed using Prism (Graphpad Software Inc.), one-way ANOVA with Tukey's multiple comparisons post-test was used to compare multiple independent groups. Mean values and SEM are indicated. ****p < 0.0001; **p < 0.01; *p < 0.05; non-significant differences not shown.
which are known to respond to IL-33 and contribute IL-13 and IL-5 to the early allergic response, but noted no overt effect of Hp-TGM on their expression by flow cytometry (Supplementary Figure 3B,C).

Hp-TGM is protective in a T cell-dependent model of airway allergy
Because allergy is primarily mediated by a type 2 adaptive immune response, we then tested Hp-TGM in a T cell-dependent model of airway sensitization and challenge, using HDM extract [60], with intraperitoneal administration of Hp-TGM during allergen sensitization (day 0) or at each day of challenge (day 7-11) (Figure 3a). In this context, a single exposure to Hp-TGM on the day of sensitisation was sufficient to strongly inhibit total cellular influx into the BALF when assessed 4 days after the final challenge dose (Figure 3b). A substantial reduction in eosinophilia was seen although not reaching statistical significance (Figure 3c). In the lung, while total cellularity did not differ greatly (Figure 3d), eosinophilia (Figure 3e,f) was strongly down-modulated in animals receiving Hp-TGM at the time of sensitization.
Levels of type 2 cytokines in lung homogenates on day 15 were then analysed. Hp-TGM administration strongly inhibited IL-13 responses in animals receiving HDM (Figure 3g), while other cytokines (IL-4, IL-5 and eotaxin-1) were not significantly induced by HDM in these experiments and no significant differences in these cytokines were observed with Hp-TGM administration (data not shown). As Hp-TGM is a potent inducer of CD25 + Foxp3 + regulatory T cells, we also evaluated their levels in the lungs of mice in these experiments. As shown in Figure 3h, the proportion of Foxp3 + Tregs differed only slightly between the groups, although compared to naïve controls there was a significant increase in mice receiving Hp-TGM on the day of allergic sensitization with HDM.

Hp-TGM sharply reduces allergic eosinophilia in response to allergen challenge
To further investigate the potential of Hp-TGM as a therapeutic option for airway allergy, we next tested its ability to dampen allergic inflammation in previously sensitized animals. Mice were sensitized with HDM at day 0 and challenged with the same allergen for five consecutive days from day 7 to day 11 after sensitization. On the same day of each HDM challenge, mice received either Hp-TGM or PBS i.p. (Figure 4a). Four days following the final challenge, total cell influx, lymphocyte infiltration and eosinophilia were measured in the BALF and lung.
In mice that received Hp-TGM, total cell numbers were sharply reduced in the BALF (Figure 4b) with a highly suppressive effect on eosinophilia (Figure 4c). In the lung, although the total cellularity was not greatly reduced (Figure 4d), a strong inhibition of eosinophilia was again evident (Figure 4e

Suppression of eosinophilia by systemic
Hp-TGM is associated with significant reduction of eotaxin-1 and IL-4 responses, rather than induction of regulatory responses We then examined if the effect of Hp-TGM during allergen challenge was accompanied by a modulation of type 2 cytokines in the lung tissues. Most notably, eotaxin-1 ( Figure 5a) and IL-4 ( Figure 5b) were significantly attenuated in Hp-TGM-treated mice, although at this time point IL-5 ( Figure 5c) and IL-13 ( Figure 5d) were not different between groups. An increase in CCL17 chemokine levels in HDM-allergic mice was similar irrespective of Hp-TGM administration (Figure 5e). We also examined mRNA transcript levels for these and other cytokines, finding no effect on eotaxin-1, IL-4 (Supplementary Figure 5A,B), or IL-5 (Figure 5f), but a significant decrease in IL-13 transcripts following Hp-TGM treatment (Figure 5g). Of note, no differences in BALF cytokines were observed (data not shown). We also measured GM-CSF and IL-3 protein levels in lung homogenates, however, no effect of Hp-TGM were observed on these molecules (Supplementary Figure 5C,D). In addition, we investigated if Hp-TGM effect on allergic eosinophilia involved induction of Tregs and their regulatory cytokines, since we and others have previously shown their importance in the suppression of airway allergy [36,61,62]. However, the percentage of Tregs in CD4 + T cells (Figure 5h) and levels of Foxp3 mRNA (Figure 5i) were only marginally increased following parenteral Hp-TGM and did not reach significance. Similarly, IL-10 protein and mRNA transcript levels (Supplementary Figure 5E,F), and TGFβ mRNA levels (Supplementary Figure 5G) were unchanged by Hp-TGM administration. Interestingly, HDM treatment resulted in a significant reduction in IL-4Rα transcripts compared to both control and Hp-TGM-treated mice (Supplementary Figure 5H), which may represent receptor downregulation following a higher level of activation.
Hp-TGM also reduces airway eosinophilia in the ovalbumin allergy model Although ovalbumin-induced models are less clinicallyrelevant than airway allergen models, since sensitization is achieved by intraperitoneal administration of OVA  protein emulsified in alum, they are characterized by a Th2 response with IL-4, IL-5, and IL-13 production, and are independent of epithelial-derived alarmin cytokines, such as IL-33 [1]. To check if parenteral Hp-TGM could also control eosinophilia in this allergy model, we administered a pre-mixed OVA/Alum solution intraperitoneally to mice at day 0, and recalled responses to OVA by administering it intranasally from day 7 to day 9 following injections with Hp-TGM 3 h earlier on the same day. On day 10, mice were culled, and airway tissues harvested (Figure 6a). In this setting, Hp-TGM significantly decreased CD45 + cell influx in the BALF (Figure 6b) and almost completely abolished eosinophilia (Figure 6c). In the lung, cellularity was little changed (Figure 6d) and while there was a strong trend for reduction of eosinophilia in the presence of Hp-TGM, this did not reach significance (Figure 6e). In addition, IL-4 and IL-13 were significantly reduced in lung homogenates of mice receiving Hp-TGM, while IL-5 and eotaxin-1 responses were little affected (Figure 6f-i). No significant differences in CCR3 (eotaxin-1 receptor) expression were seen between the different groups (Supplementary Figure 6A,B). Furthermore, no significant changes were seen in the profile of lung Foxp3 + Tregs as a results of Hp-TGM treatment, either in terms of frequency ( Figure 6j) or induction of the activation marker CD69 within the Treg population (Figure 6k). Taken together, these data support the hypothesis of a regulation of T cellmediated allergic airway eosinophilia by Hp-TGM via modulation of IL-4 and IL-13 responses.

DISCUSSION
Helminth parasites are frequently associated with protection from allergic reactivity [40,42,63,64]. Although deliberate infection with helminths has been trialled for the alleviation of inflammatory symptoms [65][66][67], future helminth-derived therapies will require defined products which can be individually evaluated as potential pharmacological agents [45]. In this report, we show that the helminth-derived TGF-β mimic, Hp-TGM, potently suppressed allergic eosinophilia in three different experimental models of airway inflammation, in each case revealing specific cellular and cytokine modulation occurring in vivo. In the first model, we appraised short-term innate responses 24 h following A. alternata exposure. Eosinophilia was sharply reduced by Hp-TGM co-administration, indicating direct effect(s) on innate cell populations, together with overall diminution of type 2 cytokines, in particular BALF IL-5 and lung IL-33, the latter known to be an essential driver of immune responses in this model [68][69][70]. In mice, IL-33 is predominantly released by stromal cells in the lung tissue, including epithelial cells, fibroblasts, and endothelial cells [71,72], while in mast cells and dendritic cells IL-33 can be induced under inflammatory conditions [73,74]. Ongoing studies aim to determine which of these populations may be down-regulated by Hp-TGM in this mode. Lung ILC2s release IL-5 and IL-13 in response to IL-33 [75,76], but were not significantly affected by Hp-TGM. One cell type that also produces IL-5 and IL-13 in response to IL-33 is the mast cell [77], and as TGF-β has been shown to suppress IL-33-induced mast cell function in the context of asthma [78], this population is a prime candidate for further investigation.
We next tested Hp-TGM effects in airway allergy models where type 2 adaptive immune responses are involved and found significant protection when Hp-TGM was administered systemically. Suppression of eosinophilia was marked, both when Hp-TGM was given at the time of sensitization, or during repeated challenge, and again we found significant inhibition of eotaxin-1, IL-4 and/or IL-13 responses, depending on the model and the time-points examined. Similar effects on eosinophils were observed with HDM, or the model antigen Ovalbumin (OVA). However, in none of the protocols did Hp-TGM particularly affect Th2 and ILC2 cell numbers, and its effects on Foxp3 + regulatory T (Treg) cells were modest at best. This is in accordance with previous data from our group showing that administration of HES in allergic asthma models suppressed airway eosinophilia without inducing Treg responses [49].
Eotaxin-1 and IL-4 are important cytokine in eosinophil migration to inflamed sites. Indeed, IL-4 has been shown to induce the expression of adhesion molecules ICAM-1 (intercellular cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) that allow eosinophils to exit the inflamed airways [79,80], as well as to promote eosinophil chemotaxis by inducing eotaxin-1 in epithelial cells. We previously reported reduction of eotaxin-1 in allergy models in the presence of HES [49], which we now suggest may be mediated by Hp-TGM.
The source of IL-4 cytokine targeted by Hp-TGM in our models remain unknown, however, T follicular helper cells (Tfh) may well be the primary target of Hp-TGM since they are the main producer of IL-4 in allergic asthma [70,81,82]. We have yet to investigate the possible involvement of other immune cells known to produce IL-4 in the context of asthma, such as basophils [83][84][85][86][87] or activated ILC2s [70]. Notably, we found that Hp-TGM not only reduced lung IL-4 cytokine levels, but also downregulated IL-4Rα gene expression to baseline. Within the T cell population, which drives the response to HDM, IL-4Rα downregulation is induced by TCR engagement which renders the cell unresponsive to IL-4 [88]. Our data therefore suggest that Hp-TGM may limit Th cell antigenic stimulation, as part of a wider picture in which administration of HES in allergy models reduces Th2 responses without skewing responses towards Th1 cells [49].
Finally, in all models tested, Hp-TGM could have affected the eosinophil population directly. Indeed, TGFβ has been shown to downregulate eosinophil responses by various means, for example, by inducing their apoptosis [22,27,28], limiting their differentiation from bone marrow precursors, limiting their proliferation by antagonizing the effect of IL-3, IL-5 and GM-CSF [19,22,89,90], by limiting their activation via downregulation of the eosinophils activation marker HLA-DR [91], or by retaining them in noninflamed tissues by enhancing expression of the chemokine receptor CXCR4 [22,92]. Although we did not directly compare the effect of Hp-TGM to TGF-β, we have previously compared the effect of TGF-β and HES in similar asthma models [49,93]. While intravenous recombinant TGF-β was shown to suppress development of Th2-mediated pathology, including airway eosinophilia the effect was mediated by the induction of Tregs [93]. Intranasal TGF-β did not show any suppression of eosinophilic BALF nor modulated Th2 cytokines [49]. In our models, systemic Hp-TGM did not significantly expand Treg frequencies nor modulated IL-3, IL-5 or GM-CSF responses. Thus, it is possible that Hp-TGM could have a different mode of action than TGF-β to control airway eosinophilia.
In summary, we present data showing that Hp-TGM administration can recapitulate generalized anti-allergic effects that were previously found for the complex mixture of H. polygyrus ES products (HES) [49,93]. Airway epithelial cells are likely to be central in Hp-TGM effect on airway eosinophilia since Hp-TGM reduced the levels of IL-33 cytokine in the Alternaria model and eotaxin-1 levels in the HDM model, both of which are primarily epithelial-derived and underpin eosinophil accumulation in the allergic lung [70]. To our knowledge this is the first time that a parasite-derived protein has been described to reduce airway allergic eosinophilia on allergen rechallenge by acting on epithelial-derived cytokines and IL-4 responses rather than on the classical IL-5 cytokine response. We believe that Hp-TGM may contain possible preventive or therapeutic potential for human allergic airway disease, as targeting eosinophil infiltration in the lung has already proven to be successful in controlling type 2-high asthma in clinical trials [3,4,8,9,[11][12][13]. Future work will explore this possibility in depth.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.