DOI: 10.5061/dryad.ncjsxkt2p
title: Data from: Repeated sensitization of mice with microfilariae of Litomosoides sigmodontis induces pulmonary eosinophilia in an IL-33-dependent manner

description: Background: Eosinophilia is a hallmark of helminth infections and
 eosinophils are essential in the protective immune responses against
 helminths. Nevertheless, the distinct role of eosinophils during parasitic
 filarial infection, allergy and autoimmune disease-driven pathology is
 still not sufficiently understood. In this study, we established a mouse
 model for microfilariae-induced eosinophilic lung disease (ELD), a
 manifestation caused by eosinophil hyper-responsiveness within the lung.
 Methods: Wild-type (WT) BALB/c mice were sensitized with dead
 microfilariae (MF) of the rodent filarial nematode Litomosoides
 sigmodontis three times at weekly intervals and subsequently challenged
 with viable MF to induce ELD. The resulting immune response was compared
 to non-sensitized WT mice as well as sensitized eosinophil-deficient
 dblGATA mice using flow cytometry, lung histology and ELISA. Additionally,
 the impact of IL-33 signaling on ELD development was investigated using
 the IL-33 antagonist HpARI2. Results: ELD-induced WT mice displayed an
 increased type 2 immune response in the lung with increased frequencies of
 eosinophils, alternatively activated macrophages and group 2 innate
 lymphoid cells, as well as higher peripheral blood IgE, IL-5 and IL-33
 levels in comparison to mice challenged only with viable MF or PBS. ELD
 mice had an increased MF retention in lung tissue, which was in line with
 an enhanced MF clearance from peripheral blood. Using eosinophil-deficient
 dblGATA mice we demonstrate that eosinophils are essentially involved in
 driving the type 2 immune response and retention of MF in the lung of ELD
 mice. Furthermore, we demonstrate that IL-33 drives eosinophil activation
 in vitro and inhibition of IL-33 signaling during ELD induction reduces
 pulmonary type 2 immune responses, eosinophil activation and alleviates
 lung lacunarity. In conclusion, we demonstrate that IL-33 signaling is
 essentially involved in MF-induced ELD development.
description:   Microfilariae purification
 MF were purified as described previously [20]. In short, cotton
 rats (<em>Sigmodon hispidus)</em> naturally infected with
 <em>L. sigmodontis </em>were bled into EDTA tubes (Sarstedt AG
 & Co. KG, Nümbrecht, Germany). The blood was diluted 1:2 in
 pre-warmed RPMI-1640 media (Gibco, ThermoFisher Scientific, California,
 USA) and layered on top of a 25% and 30% sucrose gradient (Carl Roth,
 Karlsruhe, Germany) of 0.25 M in iso-osmotic percoll (Merck, Darmstadt,
 Germany). The gradient was centrifuged at 300g for 30 min without breaks.
 The white layer containing the MF was collected and washed 3 times with
 pre-warmed RPMI-1640. MF were counted in a Neubauer chamber (Laboroptik
 GmbH, Friedrichsdorf, Germany) and adjusted to 1x
 10<sup>5</sup> in 150 µl PBS (Dulbecco´s PBS, Gibco,
 ThermoFisher Scientific, California, USA). The MF were checked for
 viability via microscopy. Bone marrow-derived
 eosinophil culture Bone marrow was isolated from hind
 legs of WT BALB/c mice using a 20-gauge needle and 5 ml RPMI-1640 for each
 bone. Isolated cells were centrifuged (400g, 10 min, 4°C) and red blood
 cells were lysed by adding 1 ml of red blood cell lysis buffer
 (eBioscience, ThermoFisher Scientific, California, USA) for 5 min at room
 temperature (RT). Cells were washed with 9 ml PBS (Gibco, ThermoFisher
 Scientific, California, USA) and centrifuged at 400g at room temperature
 for 10 min, adjusted to 1x 10<sup>6</sup>/ml in RPMI-1640 with
 the addition of 20% fetal calf serum, penicillin (10000 U/ml),
 streptomycin (100 mg/ml), glutaMAX, gentamycin (50 mg/ml) (Gibco,
 ThermoFisher Scientific, California, USA for all contents). Cells were
 cultured with 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L) and 100
 ng/ml stem cell factor (SCF) (PeproTech Inc., ThermoFisher Scientific,
 California, USA) for 4 days and subsequently with 20 ng/ml IL-5 (PeproTech
 Inc., ThermoFisher Scientific, California, USA) until day 12. Half of the
 medium was replaced on day 2, 6, 10 and the whole medium was replaced on
 day 4 and 8. Cells were adjusted using CASY automated cell counter (OLS
 OMNI Life Science GmbH & Co. KG, Bremen, Germany). Cells were
 centrifuged and adjusted to 1x 10<sup>6</sup>/ml in the
 aforementioned culture media with 10% fetal calf serum. Cell purity was
 determined using flow cytometry and cell viability was assessed using the
 Annexin-V-PI staining kit (ThermoFisher Scientiffic, California, USA) and
 was above 80% purity and 55-85% viability (S8A, B Fig).
 Sensitization and induction of ELD mice The
 sensitization protocol was adjusted from Egwang and Kazura, 1990 [7]. Mice
 were injected subcutaneously into the neck fold with 1x 10<sup>5
 </sup>dead (killed by freezing) MF in 100 µl PBS once a week for
 three weeks. Two weeks later, mice were injected with 1x 10<sup>5
 </sup>freshly isolated, living motile MF in 150 µl PBS (Dulbecco´s
 PBS, Gibco, ThermoFisher Scientific, California, USA) intravenously.
 Control mice received 150 µl PBS. HpARI2 treatment of
 ELD mice Full length HpARI2 with a 6-His tag was
 expressed in Expi293 cells, and purified by nickel affinity
 chromatography, as previously described [57, 58]. HpARI2 was used to
 inhibit IL-33 signaling during ELD development. Therefore, 10 µg HpARI2 in
 10 µl PBS (Gibco, ThermoFisher Scientific, California, USA) was
 administered intranasally under anesthesia induced with 2% isoflurane
 (Piramal Critical Care, New-Delhi, India). For ELD induction, mice were
 sensitized as usual once a week for three weeks total with dead MF and two
 weeks later, starting 1 h before the final MF challenge, mice were treated
 every three days with HpARI2. Control mice received 10 µl PBS
 intranasally. Broncho-alveolar lavage
 After euthanasia of the animals, the thorax was opened carefully
 to allow the lungs to dilate completely. The throat of the animals was cut
 open to expose the trachea. A 20G Vasofix (B. Braun Melsungen, Melsungen,
 Germany) intravenous vein catheter was inserted into the trachea and the
 lung was flushed with 1 ml PBS. The first ml was centrifuged at 400g for
 10 min at 4°C and the supernatant was used for the analysis of cytokines.
 The lung was flushed with additional 4 ml PBS (Gibco, ThermoFisher
 Scientific, California, USA). Finally, 1 ml of PBS was injected into the
 lung to dilate the lobes before paraffin (Carl Roth, Karlsruhe, Germany)
 fixation. Flushed out BAL cells were centrifuged at 400g for 10 min at 4°C
 and used for flow cytometry. Lung tissue
 dissociation The lung tissue was dissociated using the
 gentleMACS in combination with the tissue dissociation kit (Miltenyi
 Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s
 protocol. Briefly, the lung was cut into the respective lobes and added
 into the dissociation tube with 2.4 mL of 1× buffer S, 100 μL of enzyme D,
 and 15 μL of enzyme A. Lung lobes were dissociated at 37°C for 30 min
 under rotation of the tube (program: 37C_m_LDK_1) in the gentleMACS
 (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated cells were
 collected by centrifugation (400g, 10 min at 4°C) and red blood cells were
 lysed for 5 min at RT, washed and resuspended in culture media
 (eBioscience, ThermoFisher Scientific, California, USA). Finally, cells
 were filtered through a 70 µm MACS SmartStrainer (Miltenyi Biotec,
 Bergisch Gladbach, Germany). Cell viability was assessed using DAPI
 staining after lung dissociation (S8C Fig).
 Purification of native proteins from the lung tissue
 Lung tissue was placed into RIPA-buffer (eBioscience,
 ThermoFisher Scientific, California, USA) and lysed in a Precellys24 (VWR
 international GmbH, Langenfeld, Germany). Cells were incubated for 1 h at
 4°C and centrifuged for 30 min at 16,000 g at 4°C. The supernatant was
 collected and adjusted to 100 µg/ml using Bradford protein quantification.
 Samples were diluted 1:100 in Bradford reagent (Sigma-Aldrich, MERCK,
 Darmstadt, Germany) and compared to a BSA standard.
 Detection of proteins from lysed lung tissue
 10 µg lung protein extraction were used for the respective ELISA
 kits. CCL5, CCL11 (R &D Systems, Minnesota, USA), Amphiregulin
 (ThermoFisher Scientific, California, USA), major basic protein
 (Antikörper-Online, Aachen, Germany), MMP activity assay (Abcam,
 Cambridge, United Kingdom) and eosinophil peroxidase (Lifespan
 Biosciences, Washington, USA) ELISA measurements were performed according
 to the manufacturers’ protocols. Spleen
 dissociation Spleens were isolated from the euthanized
 mice and dissociated using a syringe stamp and pressing the organ through
 a 70 µm MACS SmartStrainer (Miltenyi Biotec, Bergisch Gladbach, Germany).
 Splenocytes were collected by centrifugation at 400g at RT for 5 min and
 red blood cells were lysed (eBioscience,Thermofisher Scientific,
 California, USA). Cells were washed with RPMI1640 media (Gibco,
 ThermoFisher Scientific, California), counted and used for flow
 cytometry. Microfilariae quantification from
 lung Lung and heart were isolated from the mice and
 placed into a glass petri dish. 20 G needles (B. Braun, Melsungen,
 Germany) were placed into the left and right atrium. An additional needle
 was placed into the right atrium and attached to a 10 ml syringe (B.
 Braun, Melsungen, Germany) containing PBS (Gibco, ThermoFisher Scientific,
 California, USA). The lungs were flushed with 10 ml PBS. The flow through
 was collected in a 15 min falcon tube and centrifuged for 10 min at 400g
 at RT. The supernatant was discarded and 1 ml of 1x red blood cell lysis
 buffer (ThermoFisher Scientific, California, USA) was added to the pellet.
 After 5 min at RT, the samples were centrifuged again at 400g for 10 min
 at RT. 950 µl of the supernatant was carefully removed. The pellet was
 resuspended in the remaining 50 µl and MF were counted via
 microscopy. Microfilariae quantification from
 peripheral blood 50 µl of blood was collected from mice
 in EDTA Tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany) and
 transferred into 1.5 ml Eppendorf tubes (Eppendorf SE, Hamburg, Germany).
 1 ml of red blood cell lysis (ThermoFisher Scientific, California, USA)
 was added and incubated for 5 min at RT. Following centrifugation at 400g
 for 10 min at RT, 1 ml of supernatant was removed, the pellet resuspended
 and all MF contained in the remaining 50 µl pellet were counted via
 microscopy. Flow cytometry
 10<sup>6</sup> cells were blocked in 1 µg/ml rat
 IgG/PBS (Sigma-Aldrich, MERCK, Darmstadt, Germany) for 1 h at 4°C. The
 blocking buffer was removed by centrifugation (400g, 8 min, 4°C) and the
 cell pellet was dissolved in 100 µl fixation buffer (BioLegend,
 California, USA). Cells were fixated at RT for 15 min and washed with PBS
 (Gibco, ThermoFisher Scientific, California, USA), centrifuged at 400g for
 10 min at RT and stained in 20 µl surface marker antibody mix (S1 Table)
 for 30 min at 4°C. Stained cells were washed twice with PBS and
 centrifuged at 400g for 10 min at RT. For additional intracellular
 stainings, cells were washed twice with 100 µl permeabilization buffer
 (BioLegend, California, USA) and subsequently stained for 30 min at 4°C.
 Finally, cells were washed twice with PBS and measured using a CytoflexS
 flow cytometer (Beckman Coulter, California, USA). ILC2s were gated as
 CD45<sup>+</sup>, linage<sup>-</sup>,
 TCRb<sup>-</sup>, CD90.2<sup>+</sup>,
 ST2<sup>+</sup>, GATA3<sup>+</sup>, RELM-α
 positive macrophages as CD45<sup>+</sup>,
 CD206<sup>+</sup>, Siglec-F<sup>+</sup>,
 RELM-α<sup>+</sup> and eosinophils as
 CD45<sup>+</sup>, CD11c<sup>-</sup>,
 Siglec-F<sup>+</sup>, CD11b<sup>+</sup>. Further,
 neutrophils were gated as CD45<sup>+</sup>,
 Ly6G<sup>+</sup>, alveolar macrophages as
 CD45<sup>+</sup>, CD206<sup>+</sup>,
 Siglec-F<sup>+</sup>. The gating strategy is shown in S9
 Fig   Enzyme linked immunosorbent
 assay Invitrogen ELISA (IFNγ, IL-4, IL-5, IL-33,)
 (Invitrogen, ThermoFisher Scientific, California, USA) were performed
 according to the manufacturer’s protocol from serum, bronchoalveolar
 lavage (BAL), pleura wash or lung homogenates. Plates were coated with 1x
 coating buffer. 50 µl serum was diluted 1:2 in assay buffer, 100 µl of
 either BAL, pleura wash or lung homogenates was transferred onto the
 plates alongside the respective standard samples and incubated for 2 h at
 RT and 200 rpm. Samples were blocked in assay diluent. Plates were washed
 and incubated with the detection antibody for 1 h at RT and 200 rpm.
 Plates were washed and horseradish peroxidase--conjugate was added for 30
 min at RT and 200 rpm. Colorimetric detection was performed by adding 50
 µl 3,3´5,5`-Tetramethylbenzimidin into each well. The reaction was stopped
 using 50 µl of 1 M H<sub>2</sub>SO<sub>4</sub>
 (Carl Roth, Karlsruhe, Germany) to each well. Optical density of the
 plates was measured in a SpectraMax Molecular Devices LLC, California,
 USA) at 450 and 570 nm wave length. Concentrations were calculated using
 the Softmax Pro software (Molecular Devices LLC, California,
 USA). Detection of IgE
 Parasite-specific serum IgE was detected by coating ELISA plates
 (Sigma-Aldrich, MERCK, Darmstadt, Germany) with 20 µg/ml female
 <em>L. sigmodontis </em>adult worm crude extract diluted in
 PBS (Gibco, ThermoFisher Scientific, California, USA) for 24 h at 4°C. The
 supernatant was discarded and plates were washed and blocked for 1 h with
 5% BSA/PBS (MERCK, Darmstadt, Germany) at RT. Serum samples were diluted
 1:50, 50 µl were added onto the plate and incubated for 2 h at RT. After
 the incubation, the samples were discarded and the plates were washed
 three times. The biotinylated anti-mouse-IgE antibody was diluted 1:200
 (2µg/ml) in PBS, added to each well and incubated for 1 h at RT, at 200
 rpm. Following a washing step, the plates were incubated with the
 conjugated horseradish peroxidase for 30 min at RT, shaking at 200 rpm.
 Colorimetric detection was performed by adding 50 µl
 3,3´5,5`-Tetramethylbenzimidin into each well. The reaction was stopped
 using 100 µl of 1 M H<sub>2</sub>SO<sub>4</sub>
 H<sub>2</sub>SO<sub>4</sub> (Carl Roth, Karlsruhe,
 Germany) to each well. Optical density of the plates was measured in a
 SpectraMax (Molecular Devices LLC, California, USA) at 450 and 570 nm wave
 length. DNA quant DNA quantification
 was performed as described previously [20]. In brief, the Invitrogen
 Quant-iT dsDNA Assay Kit, high sensitivity (ThermoFisher Scientific,
 California, USA), was used for DNA quantification of supernatant of
 stimulated bone marrow-derived eosinophils. Eosinophils were cultured in
 96-well plates with medium containing murine IL-5 (20ng/ml PeproTech inc.,
 ThermoFisher Scientific, California, USA). After 24 h, 2.5 U/well of the
 micrococcus exonuclease (PeproTech inc., ThermoFisher Scientific,
 California, USA) was added to all samples at 37°C for 15 min to
 disassociate DNA bound to the wells. The micrococcal nuclease reaction was
 stopped by adding 1 mM EDTA. Culture plates were subsequently centrifuged
 at 400g for 8 min at 4°C and the supernatant was removed and transferred
 to a new 96-well plate (Sigma-Aldrich, MERCK, Darmstadt, Germany). The
 quantification was done with 100 μl Quant-iT™ dsDNA HS buffer after a
 1:200 dilution. The diluted DNA Quant-iT solution was added to 20 μl of
 the supernatant. For quantification, the provided standard ranging from
 0-10 ng/ml was used. The absorption of sample and standard was measured
 using the Tecan infinity M200 (Tecan Group, Männedorf, Switzerland) at
 485/525 nm. Lung histology Mice were
 sacrificed and the lung lobes were inflated with 1 ml PBS (Gibco,
 ThermoFisher Scientific, California, USA) via the trachea using a 20G
 Vasofix intravenous vein catheter (B. Braun, Meisungen, Germany). To
 maintain the inflated status of the lung tissue, the trachea was closed
 with a yarn tied into a knot. The lung was removed together with the heart
 and placed into 2% methanol free formalin (ThermoFisher Scientific,
 California, USA) for 24 h at RT. The lung tissue was washed twice with PBS
 (Gibco, ThermoFisher Scientific, California, USA) and dehydrated by being
 submerged in ethanol baths of ascending concentration (50, 70, 80, 96,
 100%) and finally 100% xylene for 30 min (STP-120 automated sample
 processor, Especialidades Médicas Myr, S.L., Tarragona, Spain). The tissue
 was embedded in paraffin (Merck, Darmstadt, Germany) at 56°C (modular
 tissue embedding center, Especialidades Médicas Myr, S.L., Tarragona,
 Spain). Hardened paraffin blocks were cut into 4 µm sections, paraffin was
 removed with alcohol baths, sections were stained with Mayer´s hematoxylin
 and eosin (Merck, Darmstadt, Germany) and mounted with entellan (Merck,
 Darmstadt, Germany). Stitched histology images were taken on the
 Axio-observer 5 (Zeiss, Oberkochen, Germany) using Zen V3.6 (Zen Blue
 edition V3.6, Zeiss, Oberkochen, Germany). Fifty 10x10 µm Random regions
 of interest were generated using KNIME (https://www.knime.com/ [59]) and
 lacunarity analysis was
 performed using the ImageJ [60] plugin FracLac (Karperien, A., FracLac for ImageJ. http://rsb.info.nih.gov/ij/plugins/fraclac/FLHelp/Introduction.htm). Protocol adapted from Chenery et al., 2019 [61].
description: # Repeated sensitization of mice with microfilariae of Litomosoides
 sigmodontis induces pulmonary eosinophilia in an IL-33-dependent manner
 [https://doi.org/10.5061/dryad.ncjsxkt2p](https://doi.org/10.5061/dryad.ncjsxkt2p) ## Description of the data and file structure Data is contained in files in accordance to the respective figure names. For instance: the file "figure 1" contains the raw data for all graphs within Fig.1 of the manuscript. Further discription: Figure 1 contains data of the earliest ELD timepoint investigated (24h after challenge) 1\) Microfilariae retention within the lung tissue and peripheral blood 2\) Cells per gram lung tissue 3\) flow cytometry data of ILC2, Eosinophils, realm alpha positive macrophages, neutrophils Figure 2 contains serum and Lung ELISA data of the earliest timepoint investigated (24h after challenge) 1\) 2A: IL-5, 2B: IFNy, 2C: total IgE, 2D: parasite-specific IgE 2\) MMP activity assay, MPO, EPO, CCL11, CCL5 and Amphiregulin concentrations in the lung tissue Figure 3 contains data of the late ELD timepoint investigated (10d after challenge) 1\) Microfilariae retention within the lung tissue and peripheral blood 2\) Cells per gram lung tissue 3\) flow cytometry data of ILC2, Eosinophils, realm alpha positive macrophages, neutrophils Figure 4 contains the flow cytometry activation measurements of the markers CD11b, CD86, SiglecF and ST2 of lung eosinophils at day 10 post challenge. Figure 5 contains serum ELISA data from 10 days post challenge of the cytokines IL-33 and IL-4, BAL DNA content and parasite-specific and total IgE. Figure 6 contains the surface expression of the activational markers CD54, SiglecF, ST2, CD101, MHC2, CD11b, CD86, CD107 and DNA release of eosinophils after 24h in vitro culture with different concentrations of IL-33. Figure 7 contains the same data sets as figure 1 and 3 but after a treatment with the IL-33 inhibitor HpARI. It additionally contains the surface expression/activation status of lung eosinophils assessed by expression of CD86, CD11b, SiglecF and CD101. Figure 8 contains the lacunarity assesment of the lung tissue. Figure 9 contains the MMP activity assay, MPO, EPO, CCL11, CCL5 and Amphiregulin concentrations in the lung tissue. Supplement figure 1 (S1) contains MF counts of the peripheral blood 1 h after challenge. Supplement figure 3 (S3) contains spleen cell numbers, spleen eos frequencies, BAL eosinophil numbers and eosinophil activation Supplement figure 4 (S4) contians serum levels of INFy, IL-5 and total as well as parasite-specific IgE Supplement figure 7 (S7) contians serum levels of INFy, IL-5 and normalized numbers of neutrophils and alveolar macrophages within the lung Supplement figure 8 (S8) contains eosinophil purity and viability after culture for 24 and cell viability after lung dissociation. The folder randomROI contains information and the workflow used to generate random ROIs using KNIME. ## Sharing/Access information Please contact [Benjamin.lenz@ukbonn.de ](mailto:Benjamin.lenz@ukbonn.d)for additional information. ## Code/Software KNIME Random ROI generator workflow is uploaded as a file. Please folder see RandomROs