October IL-33-binding HpARI family homologues with divergent eûects in suppressing or enhancing Type 2 immune responses Florent Colomb 10 11 3 7 8 9 Adefunke Ogunkanbi 10 11 2 3 7 8 9 Abhishek Jamwal 0 10 11 5 7 8 9 Beverley Dong 1 10 11 7 8 9 Rick M. 10 11 7 8 9 Maizels 10 11 6 7 8 9 Constance A. M. Finney 1 10 11 7 8 9 James D. Wasmuth 10 11 4 7 8 9 MaEhew Higgins 0 10 11 5 7 8 9 Henry J. 10 11 7 8 9 McSorley 10 11 3 7 8 9 Alberta 10 11 7 8 9 Canada. 10 11 7 8 9 . Department of Biochemistry, University of Oxford , South Parks Road, Oxford, OX1 . Department of Biological Sciences, Faculty of Science, University of Calgary , Calgary . Department of Biology, University of York , Wentworth Way, YO10 5DD, York, United . Division of Cell Signalling and Immunology, School of Life Sciences, University of . Faculty of Veterinary Medicine, University of Calgary , Calgary, Alberta , Canada . Kavli InsYtute for Nanoscience Discovery, Dorothy Crowfoot Hodgkin Building . Wellcome Centre for IntegraYve Parasitology, School of InfecYon and Immunity 3QU , United Kingdom Alberta , Canada Dundee , Dow Street, Dundee, DD1 5EH , United Kingdom University of Glasgow , Glasgow , United Kingdom University of Oxford , South Parks Rd, Oxford, OX1 3QU , United Kingdom 2023 10 2023 12 31 -

Kingdom 6. Host-Parasite InteracYons Research Training Network, University of Calgary, Calgary, HpARI is an immunomodulatory protein secreted by the intesYnal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we ûnd that the H. polygyrus bakeri genome contains 3 HpARI family members, and that these have diûerent eûects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing, and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar aûnity for mouse IL-33, however HpARI3 does not block IL-33-ST2 interacYons. Instead, HpARI3 stabilises IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and disYnct funcYons, comprising a complex immunomodulatory arsenal of host-targeted proteins.

 IntroducAon

ParasiYc helminth infecYon aûects more than one billion p eople worldwide. The relaYonship between parasites and their hosts reûects co-evoluYonary adaptaYon to host protecYve immune mechanisms which results in parasites9long persistence in the host (1, 2). Excretory-secretory (ES) products released from helminths contain a wide range of molecules which can modulate the host immune system, suppressing anY-parasiYc immune responses and hence promote parasite survival (3, 4). The murine intesYnal parasite Heligmosomoides polygyrus bakeri (Hpb) is a prime example of this host-parasite dynamic. Hpb secreYons contain the Alarmin Release Inhibitor (HpARI) and Binds Alarmin Receptor and Inhibits (HpBARI), both of which block the IL-33 pathway and suppress type 2 immune responses in mice (5, 6). Hpb infects mice when larvae are ingested. These larvae rapidly penetrate the epithelium of the duodenum, and develop to adults which emerge into the gut lumen at around day 10 of infecYon, living for weeks as sexually mature adults in the small intesYne. The early phases of infecYon are associated with damage to the intesYnal epithelium as it is penetrated by migraYng infecYve larvae (7).

IL-33 is an IL-1 family alarmin cytokine which is consYtuYvely expressed by epithelial cells, where it is stored in cellular nuclei and released under condiYons of damage and necrosis (8). Upon release, the acYve mature cytokine is rapidly oxidised, rendering it unable to bind to its receptor ST2 (9), limiYng its ST2-dependent acYvity in vivo to the local milieu and to a short period of Yme aner release. IL-33 acts as an iniYator of allergic diseases, especially asthma where both IL-33 and ST2 are geneYcally linked to asthma heritability (10), and are targets for new biologic treatments (11, 12). IL-33 also plays a signiûcant role in parasite expulsion, with deûciency or blockade of the IL-33 pathway resulYng in increased parasite burden (8, 13, 14). In our previous work we idenYûed HpARI, showing that it binds IL-33 and blocks type 2 immune responses (5), and used structural studies to demonstrate how this IL-33-ST2 inhibiYon is achieved (15). Here, we show that the Hpb genome encodes a family of three HpARI proteins: HpARI1, HpARI2 and HpARI3, with the original HpARI now renamed as HpARI2. We expressed and tested these proteins for their acYvity in vitro and in vivo, and unexpectedly found that HpARI3 enhances rather than suppresses IL-33 responses.

Methods Mass spectrometry

The excretory/secretory products of Hpb (HES) was prepared as described previously (16), and a 2 ¿g sample was used for mass spectrometry analysis. Triethylammonium bicarbonate (Sigma) and dithiothreitol (Merck Millipore) were added to ûnal concentraYons of 100 mM and 10 mM, respecYvely, and sample incubated at 70¡C for 10 min. AlkylaYon was carried out by addiYon of 20 mM iodoacetamide for 30 min in the dark. Sample was incubated overnight in the presence of 50 mM dithiothreitol and 200 ng trypsin was added, and incubated overnight at 37¡C. TriûuoraceYc acid (Fisher ScienYûc) at 10% sample vo lume was added, C18 clean-up was carried out using an Empore-C18 (Agilent) solid phase extracYon cartridge, and dried samples resuspended in 1% Formic acid (Fisher Chemical).

Analysis of pepYde readout was performed on a Q ExacYve# plus Mass Spectrometer (Thermo ScienYûc) coupled to a Dionex UlYmate 3000 R S (Thermo ScienYûc). Liquid chromatography buûers used were: buûer A (0.1% form ic acid in Milli-Q water (v/v)) and buûer B (80% acetonitrile and 0.1% formic acid in Milli-Q water (v/v). An equivalent of 1.0 µg of pepYdes from each sample were loaded at 10 ¿L/min onto a µPAC trapping C18 column (Pharmaûuidics). The trapping column was washed for 6 min at the same ûow rate with 0.1% TFA and then switched in-line with a Pharma Fluidics, 200 cm, µPAC nanoLC C18 column. The column was equilibrated at a ûow rate of 300 nl/min for 30 min. The pepYdes were eluted from the column at a constant ûow rate of 300 nl/min with a linear gradient from 2% buûer B to 5.0% buûer B in 5 min, from 5.0% B to 35% buûer B in 125 min, and from 35% to 98% in 2 minutes. The column was then washed at 98% buûer B for 20 min and then washed at 2% buûer B for 20 minutes. Two blanks were run between each sample to reduce carry-over. The column was kept at a constant temperature of 50¡C.

Q-exacYve plus was operated posiYve ionizaYon mode us ing an easy spray source. The source voltage was set to 2.90 kV and the capillary temperature was 275¡C. Data were acquired in Data Independent AcquisiYon Mode as previously described (17), with liEle modiûcaYon. A scan cycle comprised a full MS scan (m/z range from 345-1155), resoluYon was set to 70,000, AGC target 1x106, maximum injecYon Yme 100 ms. MS survey scans were followed by DIA scans of dynamic window widths with an overlap of 0.5 Th. DIA spectra were recorded at a resoluYon of 17,500 at 200 m/z using an automaYc gain co ntrol target of 2x105, a maximum injecYon Yme of 100 ms and a ûrst ûxed mass of 100 m/z. Normalised collision energy was set to 27% with a default charge state set at 3. Data for both MS scan and MS/MS Data Independent AcquisiYon (DIA) scan events were acquired in proûle mode.

Mass spectrometry data analysis: Label-free analysis was performed in Maxquant (version 2.0.3.0) using the generate RAW ûles. Enzyme speciûcity was set to that of trypsin, allowing for cleavage N-terminal to proline residues and between asparYc acid and proline residues. Other parameters used were: (i) variable modiûcaYons- oxidaYon (M), protein N-acetylaYon, gln ³ pyro-glu, deamidaYon (NQ), deoxidaYon (MW); (ii ) ûxed modiûcaYons, cysteine carbamidomethylaYon; (iii) database: in-house database; (iv) MS/MS tolerance: FT-MS 10ppm, FT-MSMS 0.06 Da; (v) maximum pepYde length, 6; (vi) maximum missed cleavages, 2; (vii) maximum of labelled amino acids, 3; and (vii) false discovery rate, 1%. Match Between Runs (MBR) was set to true. Unique pepYdes were used for protein quanYûcaYon.

 Expression and Puriûca;on of HpARI1-3

Proteins were expressed and puriûed using a protocol described previously (5). Brieûy, mammalian expression constructs carrying C-terminal 6X-His tagged gene of interest (HpARI13, or ST2 ectodomain) were transfected individually into Expi293F cells (Thermo Fisher) using the Expifectamine transfecYon kit (Thermo Fisher). Cell supernatants were harvested 96 hours post-transfecYon and the expressed recombinant proteins were than captured from the ûltered supernatants using Ni-NTA chromatography.

 Surface Plasmon Resonance

To measure their binding aûniYes for mIL-33, the puriûe d HpARI variants were ûrst polished to remove aggregates, using a Superdex S75 10/300 column in 1XPBS and chemically bioYnylated using EZ-LinkTM

Sulfo-NHS-BioYn (Thermo Fisher ScienYûc) following manufacturer9s instrucYon. All SPR experiments were carried out using a Biacore T200 instrument (GE Healthcare) using a CAP chip (BioYn CAPture kit (CyYva)) in an SPR buûer containing 20 mM Tris-Cl pH 8.0, 200 mM NaCl, 1mg/ml salmon sperm DNA and 0.05% Tween20. Puriûed mIL-33 was equilibrated in the SPR buûer using a PD-5 column prior to the experiment. The sensor surface was ûrst coated with oligonucleoYde coupled streptavidin following manufacturer9s instrucYons. Individual bioYnylated HpARI variants were captured on diûerent ûow cells followed by individual injecYon s of 2-fold concentraYon series of mIL33 (50 nM to 0.195 nM for HpARI1 and HpARI2 and 200 nM to 0.781 nM for HpARI3) at a ûow rate of 40 µl/min, with 400 s associaYon Yme and 1000 s dissociaYon Yme. Aner each injecYon, the sensor chip surface was regenerated by injecYng 10 µl of 6M guanidium-HCl and 1M NaOH pH 11.0 mixed in a 4:1 raYo. All SPR data were analysed using the BIA evaluaYon sonware 2.0.3 (GE Healthcare).

 Animals

BALB/cAnNCrl and C57BL/6JCrl mice were purchased from Charles River, UK. Heterozygous IL13eGFP mice were provided by Prof Andrew McKenzie (18) and were bred in-house. Experiments were caged-blocked: each cage contained one member of each group in the experiment, thus controlling for cage eûects. Mouse accommodaYon and procedures were performed under UK Home Oûce licenses with insYtuYonal oversight performed by qualiûed veterinarians.

 In vivo Alternaria model challenge.

BALB/c mice were intranasally administered with 50 ¿g Alternaria allergen (Greer XPM1D3A25) and 10 ¿g HpARI1, HpARI2 or HpARI3 suspended in PBS, carried out under isoûurane anaesthesia. Mice were culled 24 h later and bronchoalveolar lavage (BAL) was collected (4 lavages with 0.5 ml ice-cold PBS). Lungs were taken for single-cell preparaYon and ûow cytometry, as previously described (5). Lung and BAL eosinophils were idenYûed as SiglecFhiCD11c3CD45+ live cells. Lung ILC2 were idenYûed as ICOS+CD90.2+Lineage3CD45+ cells. IL-5 levels were quanYûed in undiluted BAL ûuid by ELISA following manufacturer9s instrucYons (Invitrogen).

 CMT-64 culture and treatment

CMT-64 cells (ECACC 10032301) were maintained in complete RPMI [RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml Penicillin and 100 ¿g/ml Streptomycin (ThermoFisher ScienYûc)] at 37°C, 5% CO2. 96-well plates were seeded at 5x104 cells per well. Cells were grown to 100% conûuency, washed and incubated in complete RPMI containing HpARI2 or HpARI3 at 10 ¿g/ml or in combinaYon at various concentraYon as indicated. Cells were snap frozen on dry ice for at least 1 h, then thawed and incubated at 37°C as indicated, prior to collecYon of supernatants and applicaYon to bone marrow cell cultures.

 Bone marrow assay and ELISA

Bone marrow single cell suspensions were prepared from C57BL/6 mice, by ûushing Ybias and femurs with RPMI 1640 medium using a 21 g needle. Cells were resuspended in ACK lysis buûer (Gibco) for 5 min at room temperature, prior to resuspension in complete RPMI (with 10% FCS, 1% Penicillin/Streptomycin, 1% L-glutamine, Gibco) and passing through a 70 ¿m cell strainer. Cells were cultured in round-boEom 96-well-p lates in a ûnal 200 ¿l volume, containing 0.5×106 cells/well. IL-2 and IL-7 (Biolegend) were added at 10 ng/ml ûnal concentraYon. CMT freeze-thaw supernatant prepared as described above were added at 50 ¿L per well. Cells were then cultured at 37°C, 5% CO2, for 5 days, prior to supernatant collecYon. IL-5 and IL-13 concentraYon were assessed fol lowing manufacturer9s instrucYons using mouse uncoated IL-5 and IL-13 ELISA kits (Invitrogen).

 Assessment of ST2 binding by IL-33 in the presence of HpARI variants

Puriûed mST2 ectodomain (20 µM) was mixed individually with 40 µM of mIL33, 25 µM HpARI2:mIL33 and HpARI3:mIL33 to a ûnal volume of 200 ¿l. All samples were incubated for 1 hour on ice and then applied to the Superdex S200 10/300 GL column (cyYva) preequilibrated with buûer containing 20 mM Tris-Cl pH 8.0 and 100 mM NaCl. The eluted fracYons were analyzed by SDS-PAGE.

Sta;s;cs Data was analysed using Graphpad Prism v10.0.0. When comparing independent groups, oneway ANOVA with Dunnet9s post test was used. When comparnig groups over a Ymecourse, two-way ANOVA with Dunnet9s post test was used. Standard error of mean is used throughout. 7777 = p < 0.0001, 777 = p < 0.001, 77 = p < 0.01, 7 = p < 0.05, N.S. = Not signiûcant (p > 0.05).

 Results H. polygyrus bakeri genome encodes three HpARI-like proteins

HpARI was idenYûed by in-house transcriptomic and proteomic analysis of Hpb (5). Homology searching of published Hpb transcriptomic and genomic data via Wormbase ParaSite (19) idenYûed a family of 3 HpARI genes from Hpb, all of which have the same characterisYc domain organisaYon of a secretory signal pepYde followe d by 3 Complement Control Protein (CCP) domains (Figure 1A). Proteomic analysis of Hpb excretory/secretory products (HES) showed that the most abundant HpARI family member was HPOL_0002204701 (Intensity = 9x109), and so was named HpARI1, while the previously-idenYûed <HpARI= protein, HPBE_0000813301 (5), was renamed as HpARI2 (Intensity = 1x10 9), and the ûnal homologue with lowest intensity in HES, HPBE_0002531401, was renamed HpARI3 (Intensity = 4x10 7). Further analysis of a recent transcriptomic study of Hpb (20) showed that while HpARI1 and HpARI3 are expressed at a fairly consistent level throughout the lifecycle (albeit with HpARI3 at a far lower level compared to HpARI1), the expression of HpARI2 peaks early in infecYon (during the Yssue-dwelling phase), with similar trends in males and females for each HpARI (Figure 1B). The 3 HpARI family members show a high level of sequence similarity (69-81% idenYty between the 3 HpARIs), however the HpARI3 sequence contains a notable 3 amino acid deletion (at E94_R96 in HpARI2 sequence) at the N-terminal end of CCP2 (Figure 1A), a region which contains an HpARI2-IL-33 interaction site (15).

 The HpARI family members all bind IL-33 with sub-nanomolar aûniAes.

The 3 HpARI family members were expressed in mammalian cells and puriûed for further tesYng. Each HpARI was added to the CMT-64 cells (which express high levels of IL-33 (5)), followed by freeze-thaw necrosis-induced IL-33 release. In this assay, all 3 proteins could suppress the IL-33 signal in a dose dependent manner, indicaYng that they compete for IL-33 binding with the ELISA anYbodies used for detecYon ( Figure 2A). HpARI3 required an approximately 8-fold higher concentraYon for IL-33 suppression (IC50 of HpARI1 and HpARI2 ~8x10-10 M, HpARI3 ~6x10-9 M). When binding aûnity for IL-33 was tested by surface plasmon resonance, HpARI3 was likewise shown to have a lower aûni ty for IL-33, albeit sYll with a subnanomolar KD: HpARI1/2 have KD around 4x10-11 M, while HpARI3 has a KD of 4x10-10 M (Figure 2B).

Signal peptide

CCP1

CCP2 HpARI1 MYRLFLVLGFLTFINAAnLRCKFTDeERDKGYTGMLVnGKeRKaArNGtTVELICGRGNHNYTCtSGhLTanSlRQeRCGCDGLfEtLFDMPKEkRPSPM 100 HpARI2 MYRLFLALGFLTFINAAgqRCRFTDVERDKGYTGMLLkGRlRKTAGNGrTVELICGRGNHNYTCeSGVLkEsSpRQARCGCkGILLEEMLFFDMPKEeRRPSPM 100 HpARI3 MYRIiLtLGFLTFINAAsLgCKFTDVEKDKGYTGMLVgGKrReSAANGqkVELVCGRGNHNYTCkSGILTEeSyRQGRCGCEGILqMLlDkPK---dSPM 97 HpARI1 YsdITYDmpnMpniprVGRhGIWNGVDYRNGSvtRPYCETGPVINGSPKAVCVSGRWVPeLGDCPRMCSLSSLKEsGKFLcVnATTKGEELNPmPRlaqL 200 HpARI2 YDSVTYYDPTP-NTPTTTVVGKDGIWNGVDYYRNGSTVKPYCDTGPVINGSsKAVCVSGKWVPtLGvCPKMCSIgSLKENGKFVdVTATTKGDELNPpPRREQTLL 199 HpARI3 YDSVTYDPTP-NTPTTVGKDGIWNGVDYRNGSTVRPYCDTGPIINGSPKAVCVSGKWVPkLGDCPKMCSISSLKENGKFLnVTATTtGDELNPrPREQTL 196

CCP3 HpARI1 IPIVRnVneDeVQHrVKArAfCKAEsSTTAADhVQEFECDNGKWKPEPVPCP 252 HpARI2 IIPPIIVVRKVDKDKVQHGVKVVALCKAEdSTTAAEgVQEFECDNGKWKPEPVPCP 251 HpARI3 IPIVRKVDKDKVQHGVKVVALCKAkySTTAAEnVQkFECDNGKWKPkPVPCs 248

Day post-infection Fig 1: Sequences of HpARI family members.

A. Alignment of HpARI1, HpARI2 and HpARI3. Grey highlight indicates agreement with consensus. Signal peptide and CCP domains are indicated by red boxes, coverage of peptides identified by mass spectrometry of HES indicated by blue boxes. Residues in HpARI2-IL-33 interface highlighted in red.

B. Transcription of HpARI1, HpARI2 and HpARI3 in male and female parasites over a time course of infection. Data retrieved from the Sequence Read Archive, PRJNA750155.

CCP2

CCP3 HpARI1 females HpARI1 males HpARI2 females HpARI2 males HpARI3 females HpARI3 males bioRxiv preprint doi: https://doi.org/10.1101/20l23.10.09.561567; this version posteHdOpcAtoRberI-110, 2023. The copyright holder for this ) preprint (which was not certified by peer review2) is0the author/funder, who has granted bioRxiv a license to display the preprint in

m0 0 perpetuity. It is mad/e available under aCC-BY-NC-ND 4.H0IpntAernRatIi-o2nal license. -20

HpARI-3 0 -20

HpARI1 K (M) = 2.66x10-11

D 500

A. IL-33 detected in supernatant of CMT-64 cultures after freeze-thaw in the presence of HpARI1, HpARI2 or HpARI3.

Surface plasmon resonance (SPR) analysis of mouse IL-33 binding to chip-bound HpARI2 or HpARI3.

 HpARI family show diûering eûects on IL-33-dependent responses in vivo

To test how each HpARI aûected IL-33-dependent responses, we coadministered them intranasally to BALB/c mice with Alternaria allergen (a highly IL-33-dependent model (21-23)), and assessed type 2 innate lymphoid cell (ILC2) and eosinophil responses 24 h later (Figure 3A). HpARI2 potently suppressed BAL and lung eosinophilia, ILC2 responses and IL-5 release, as shown previously (5), while HpARI1 had a similar, albeit slightly blunted, suppressive eûect (Figure 3B-G). Surprisingly, HpARI3 had the opposite eûect, amplifying responses in this model, with signiûcantly increased lung eosinophilia and ILC2 acYvaYon (CD25 upregulaYon and increased cell size measured by FSC) as well as total IL-5 release (Figure 3B-G). These increased responses in the presence of HpARI3 are remarkably similar to that seen with a truncaYon of HpARI2 lacking the CCP3 domain (HpARI_CCP1/2), which also ampliûes IL-33 responses in vivo (14, 24). While both HpARI2 full-length protein and the HpARI_CCP1/2 truncaYon can both bind to IL-33, only HpARI2 full-length protein can block IL-33-ST2 interacYons, while HpARI_CCP1/2 cannot (24), therefore we also tested the IL-33 blocking ability of HpARI3.

 HpARI3 does not prevent IL-33 from binding to ST2

To determine whether diûerent HpARI variants can prevent IL-33 from binding to ST2, we deployed a size exclusion chromatography assay to assess IL-33-HpARI-ST2 complex formaYon (15). We ûrst conûrmed the formaYon of a complex of ST2 and IL-33 as the two proteins comigrated as a higher molecular weight complex when compared with individual components (Figure 4A). In contrast, as previously observed (15) the eluYon proûle of ST2 was unaltered in presence of HpARI2:mIL33 complex, showing that HpARI2 prevents IL-33 from binding to ST2 (Figure 4B). In contrast, HpARI3 did not prevent IL-33 from binding to ST2 as indicated by eluYon peak shin in which ST2, IL-33 and HpARI3 co-elute, showing the formaYon of a complex containing all three components (Figure 4C). Therefore, in contrast to HpARI2, HpARI3 interacts with IL-33 in a manner which does not prevent IL-33-ST2 interacYon.

 HpARI3 stabilises IL-33

c 1 1 D C SiglecF

CD25 1025 x (s20 l i ph15 o in10 s o e 5 L BA 0 C

S PB Ð I1 I2 I3

R R R pA pA pA +H +H +H

Alternaria i.n.

777 777 7777

7777

S PB Ð I1 I2 I3

R R R pA pA pA +H +H +H

Alternaria i.n.

0.00

A. Experimental setup for B-G.

B. Flow cytometry of BAL Siglecf versus CD11c, gated on CD45+ live cells, showing eosinophil gate (left). Flow cytometry of CD25 on lung ILC2, gated on live ICOS+CD90+Lin3CD45+ cells (right). Representative samples shown. C. Alternaria allergen was co-administrered with HpARI1, HpARI2 or HpARI3 as shown in A, and BAL eosinophil numbers (Siglecf+CD113CD45+) measured 24h later D. Eosinophil (SiglecfhiCD113CD45+) numbers in lung tissue.

E. CD25 expression level on lung ILC2 (ICOS+CD90+Lin3CD45+).

FSC mean in lung ILC2 (ICOS+CD90+Lin3CD45+) G. IL-5 levels in cell-free BAL fluid.

Data representative of two repeat experiments with 4 mice per group. Error bars show SEM. 0 5 1 (x 4 s l i ph 3 o in 2 s eo 1 g un 0 L

PBS +ÐHpARI+1HpARI+2HpARI3

Alternaria i.n.

77 777 AU40 (m30 e cn 20 a rb 10 o s 0 b A-10 ) 30 U A m20 ( e cn 10 a b ro 0 s b

A-10 mST2 + HpARI3:mIL33 As HpARI3 cannot block IL-33-ST2 interacYons, and instead ampliûed responses in an IL-33dependent model, we hypothesised that HpARI3 binding to IL-33 may stabilise the cytokine, extending its half-life. To test this, we used an in vitro IL-33 release and inacYvaYon assay which we developed previously (24). In this assay, CMT-64 cells are freeze-thawed to induce necrosis, and released IL-33 is incubated at 37¡C for between 15 min and 48 h aner release, to allow inacYvaYon of the cytokine. Supernatants are sub sequently applied to naïve IL13eGFP murine bone marrow cells in the presence of IL-2 and IL-7 to support ILC2 diûerenYaYon, and ILC2 acYvaYon was assessed by ûow cyto metry for acYvaYon markers (IL13eGFP and CD25 upregulaYon) and ELISA for secreYon of IL-5 and IL-13. In medium control wells, CMT-64 freeze-thaw supernatants could only induce signiûcant IL-5 and IL-13 producYon when applied within 6 h of thaw, indicaYng i nacYvaYon of IL-33 at later points of this Ymecourse, as shown previously (24). Conversely, CMT-64 cells freeze-thawed in the presence of HpARI2 could not induce any ILC2 acYvaYon, due to blockade of released IL-33.

In the presence of HpARI3 however, IL-33 in supernatants was able to sYmulate high levels of IL-5 and IL-13 release and ILC2 acYvaYon ( Figure 5B-E), indicaYng strong IL-33 responses which were not inacYvated even aner incubaYon of IL-33 -containing CMT-64 supernatants for 48 h at 37¡C.

Finally, to determine whether the eûects of HpARI2 and HpARI3 could compete in combinaYon, we added each HpARI at 1, 10 or 100 ng/ml to the assay described above, and measured released IL-5. While sole HpARI3 treatment caused a dose-dependent increase in IL-5 release, HpARI2 caused a decrease, as expected. In combinaYon, equivalent concentraYons of each HpARI showed no signiûcant change compared to medium-only control, while an excess of HpARI2 signiûcantly suppressed responses, and an excess of HpARI3 signiûcantly enhanced responses (Figure 5F). Therefore, the suppression or enhancement of IL-33 responses in vivo by HpARI2 or HpARI3 will depend on the predominance of each molecule in the local milieu. ** ******** C 15 l)m 10 / g n ( 3 1 IL 5

1 10 time post thaw (h)

Media HpARI2 HpARI3 ** 100 *** Media HpARI2

HpARI3 )100 l m / ng10 ( 3 I R 1 A p H

0 45 min

48 h

IL-13-eGFP 45 min 48 h In this study, we describe a family of 3 HpARI proteins, with high sequence similarity but diûering eûects on responses to IL-33. While HpARI1 and Hp ARI2 suppress responses to IL-33, HpARI3 lacked the ability prevent IL-33 from binding to ST2 and instead stabilised this highly labile cytokine in its acYve form, resulYng in ampliûca Yon of responses to IL-33 in in vivo and in vitro models.

The IL-33 acYvity-amplifying ability of HpARI3 was unexpected, and would seem counterintuiYve as total ST2-deûcient mice are more suscepYble to Hpb infecYon (13), implying the IL-33 pathway is largely helminth-toxic. However, addiYonally to the welldescribed type 2 immunity-inducing eûects of IL-33, recent publicaYons have described a range of eûects of IL-33 which can suppress type 2 immunity. IL-33 can acYvate and expand Foxp3+ regulatory T cells (25), and in helminth infecYon deleYon of IL-33 in myeloid cells results in a defecYve regulatory T cell response, increased type 2 immunity, and accelerated helminth ejecYon (26). Furthermore, IL-33 can acYvate na tural killer cell (27), Th1 (28) and CD8+ T cell (29) IFN-g producYon, potenYally suppressing type 2 immunity. As both Th1/CD8+ IFN-g (30, 31) and Foxp3+ regulatory T cell (32) responses have been demonstrated to control type 2 immunity in Hpb infecYon, either of these pathways could be the target of HpARI3 responses.

Although HpARI3 expression was maintained at a lower level than that of HpARI1 or HpARI2, all 3 HpARI proteins were detectable in HES. As HpARI2 and HpARI3 can compete in in vitro cultures, we hypothesise that diûerenYal Yming or localis aYon of HpARI protein release could determine the local HpARI2 / HpARI3 raYo, and therefore the resulYng response to IL-33 during infecYon. IL-339s inûammatory versus immunosuppressive eûects are parYally controlled by context of release: when IL-33 was deleted in epithelial cells, type 2 immunity and parasite ejecYon was deûcient, while when IL-33 was deleted in myeloid cells, regulatory T cell responses were reduced, resulYng in increased type 2 immunity and acerated parasite ejecYon (26). It would therefore be most advantageous for the parasite to block IL-33 in the local environment of the parasite during the early Yssue-dwelling phase of infecYon (where necroYc damage to the epithelium is most likely), while IL-33 enhancement in the draining lymph node could amplify Th2-suppressive IFN-g and regulatory T cell responses. Further work is required to invesYgate the roles of this family of immunomodulatory proteins during acYve infecYon.

Acknowledgements This work was funded by awards to HJM from LONGFONDS|Accelerate as part of the AWWA project, the Medical Research Council (MR/S000593/1) and Wellcome 221914/Z/20/Z. We would like to acknowledge the support of Cara Henderson and Alan Score of the FingerPrints Proteomics Facility at the University of Dundee, which is supported by the 'Wellcome Trust Technology Pla†orm' award [097945/B/11/Z]. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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