Il 33, by R&D Systems - Product details

1

Immunohistochemical Analysis of Wound Healing

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Cryosections (10 μm) were used for the detection of IL-33 and F4/80 (macrophages) by immunohistochemistry following procedures outlined previously,^{7 (link)} except that the following primary antibodies were used: IL-33 (R&D Systems) and F4/80 (Abcam, Cambridge, MA). Paraffin sections were used for the detection of mast cells by toluidine blue staining and Ly-6G (neutrophils; BD Biosciences, San Jose, CA) by immunohistochemical staining as described.^{7 (link), 17 (link)} For quantification of cell density, cells were counted in digital images taken in high-power fields immediately adjacent to either side of the wound and area was determined using Axiovision software (Carl Zeiss Imaging Solutions, Thornwood, NY). Cell densities (cell number per mm²) were then calculated. Cell densities for the right and left sides of each wound were averaged to obtain the mean cell density for each sample.
For immunofluorescence staining of IL-33 and GFP and immunostaining for ST2, paraformaldehyde-fixed frozen sections were used in conjunction with primary antibodies specific for IL-33 and ST2 (R&D Systems) or GFP (Abcam) and appropriate Alexa Fluor-conjugated (Thermo Fisher) or biotinylated (Vector Laboratories) secondary antibodies, using standard methods. Staining was visualized using a Nikon A1Rsi resonant scanning confocal microscope.

Wulff B.C., Pappa N.K, & Wilgus T.A. (2018). Interleukin-33 encourages scar formation in murine fetal skin wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 27(1), 19-28.

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2

Investigating IL-33 Signaling in HTNV-Infected HUVECs

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Human umbilical vein endothelial cells (HUVECs) were prepared by a previously described method [29 (link)]. The HTNV strain 76–118 and inactivated HTNV (mock virus) control were prepared and stored in our lab as described [7 (link)]. For all infections, the virus was allowed to adsorb to HUVECs at a multiplicity of infection (MOI) of approximately 1 in serum-free EGM maintenance medium for 2 h at 37°C. The cells were then washed and incubated in EGM growth medium with 10% fetal bovine serum. The proportion of infected HUVECs was tested using immunofluorescence. At 48 hours postinfection, over 90% of the HUVECs expressed viral nucleocapsid protein in the cytoplasm.
HUVECs were seeded 24 h before treatment. When the cell confluence up to 60%-70%, the cells were infected with HTNV/mock virus (MOI = 1) for 48 h or treated with interleukin-33 (IL-33, R&D system, USA) for 6 h; alternatively, the cells were pre-infected with HTNV/mock for 48 h and then treated with 20 ng/ml IL-33 for another 6 h. To assess the role of ST2 and its signalling pathways in the induction of pro-inflammatory cytokines via IL-33 stimulation, HUVECs were exposed to human recombinant sST2 (R&D Systems, USA) or inhibitors (Calbiochem, USA) of the indicated signalling pathways at different concentrations for 2 h prior to the stimulation indicated above.

Zhang Y., Zhang C., Zhuang R., Ma Y., Zhang Y., Yi J., Yang A, & Jin B. (2015). IL-33/ST2 Correlates with Severity of Haemorrhagic Fever with Renal Syndrome and Regulates the Inflammatory Response in Hantaan Virus-Infected Endothelial Cells. PLoS Neglected Tropical Diseases, 9(2), e0003514.

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Investigating IL-33 Modulation of Mast Cell Responses to Rhinovirus

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Human MCs (1 × 10⁶/mL) were incubated with IL-33 (1–10 ng/mL, R&D Systems, Abingdon, UK) for 6–24 h in a humidified 37 °C incubator with 5% CO₂ before transcriptomic and reverse transcription qPCR analyses. For HRV16 infection, MCs were pre-treated with IL-33 and then incubated with increasing multiplicity of infection (MOI: 1–7.5) of infectious virus or UV-irradiated virus (as a control) for 1 h, washed twice then resuspended in StemPro media (0.5–1 × 10⁶/mL) for specified times before harvesting. For ICAM-1 blocking experiments, LAD2 MCs or CBMCs (1 × 10⁶/mL) were treated with 10 ng/mL IL-33 for 23 h followed by 1 h with mouse anti-human ICAM-1 (clone BBIG-I1 (11C81), 10 µg/mL, R&D Systems, Abingdon, UK) or IgG2a isotype control (10 µg/mL, R&D Systems, Abingdon, UK). Experimental layouts are shown in Supplementary Materials File (Figure S1).

Akoto C., Willis A., Banas C.F., Bell J.A., Bryant D., Blume C., Davies D.E, & Swindle E.J. (2022). IL-33 Induces an Antiviral Signature in Mast Cells but Enhances Their Permissiveness for Human Rhinovirus Infection. Viruses, 14(11), 2430.

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4

Murine Cytokine Quantification via ELISA

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The release of murine TNFα, CXCL1, CXCL2/3, IL-33 (all R&D Systems), and HMGB1 (E-20749; Qayee-Bio) in the BAL of mice after LPS inhalation was determined by enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.
Additionally, we detected IL-33 and HMGB1 from the supernatants of bone marrow neutrophils under the indicated conditions. Human IL-33 (R&D Systems) and HMGB1 (LS-F4038-1; LS-Bio) were measured from the supernatants of human PMNs. Western blots intensity was measured by ImageJ (Version 1.49v; National Institute of Health; USA).

Ngamsri K.C., Gamper-Tsigaras J., Reutershan J, & Konrad F.M. (2021). Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation. Frontiers in Medicine, 8, 591790.

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5

Purification and Culture of Eosinophils

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Purified blood–derived eosinophils (>97% purity) were isolated from NJ.1638 mice as described previously.^{27 (link),28 (link)} In some experiments, bone marrow–derived eosinophils^{26 (link)} from WT mice were generated to confirm the results found with peripheral blood eosinophils. Eosinophils were either cultured in complete RPMI 1640 media for 24 hours at 2.5 × 10⁶/mL with IL-5 media (10 ng/mL IL-5; PeproTech, London, United Kingdom) to generate IL-5–treated eosinophils (IL-5 EOS) or were cultured in IL-33 Act media (10 ng/mL IL-5 with 10 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF; PeproTech] and 40 ng/mL IL-33 [R&D Systems]) to generate IL-33–activated eosinophils (IL-33 Act EOS; Fig E3, B). Cells were washed 3 times in complete RPMI 1640 media to remove cytokines before coculture experiments with ILC2s. For chemotaxis assays, cell-free supernatants were collected from eosinophils cultured for 48 hours in IL-5 media or IL-33 Act media. Controls included IL-5 media and IL-33 Act media that were not cultured with eosinophils. For adoptive transfer experiments, cultured eosinophils were washed and transferred intratracheally into the lungs of OVA-sensitized/challenged mice as described previously.^{27 (link)}

LeSuer W.E., Kienzl M., Ochkur S.I., Schicho R., Doyle A.D., Wright B.L., Rank M.A., Krupnick A.S., Kita H, & Jacobsen E.A. (2023). Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice. The Journal of allergy and clinical immunology, 152(2), 469-485.e10.

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Purification and Culture of Eosinophils

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Purified blood–derived eosinophils (>97% purity) were isolated from NJ.1638 mice as described previously.^{27 (link),28 (link)} In some experiments, bone marrow–derived eosinophils^{26 (link)} from WT mice were generated to confirm the results found with peripheral blood eosinophils. Eosinophils were either cultured in complete RPMI 1640 media for 24 hours at 2.5 × 10⁶/mL with IL-5 media (10 ng/mL IL-5; PeproTech, London, United Kingdom) to generate IL-5–treated eosinophils (IL-5 EOS) or were cultured in IL-33 Act media (10 ng/mL IL-5 with 10 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF; PeproTech] and 40 ng/mL IL-33 [R&D Systems]) to generate IL-33–activated eosinophils (IL-33 Act EOS; Fig E3, B). Cells were washed 3 times in complete RPMI 1640 media to remove cytokines before coculture experiments with ILC2s. For chemotaxis assays, cell-free supernatants were collected from eosinophils cultured for 48 hours in IL-5 media or IL-33 Act media. Controls included IL-5 media and IL-33 Act media that were not cultured with eosinophils. For adoptive transfer experiments, cultured eosinophils were washed and transferred intratracheally into the lungs of OVA-sensitized/challenged mice as described previously.^{27 (link)}

LeSuer W.E., Kienzl M., Ochkur S.I., Schicho R., Doyle A.D., Wright B.L., Rank M.A., Krupnick A.S., Kita H, & Jacobsen E.A. (2023). Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice. The Journal of allergy and clinical immunology, 152(2), 469-485.e10.

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7

Isolation and Expansion of ILC2s from Murine Lungs

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Naive WT mice were intratracheally administered 500 ng/50 μL of IL-33 (R&D Systems) in PBS on days 0 and 2 and humanely killed on day 4 to isolate lung single cell suspensions as described above. Cells were depleted of Lin⁺ cells by magnetic bead separation according to the manufacturer’s recommendations (Miltenyi Biotec, San Diego, Calif). The lineage-depleted population was sorted by flow cytometry for ILC2s (Lin⁻CD45⁺CD90.2⁺Sca-1⁺ST2⁺) on a BD Biosciences FACS Aria III SORP Cell Sorter. The purity of sorted ILC2s was >99%. ILC2s were cultured in complete RPMI 1640 with IL-2 (10 ng/mL), IL-7 (10 ng/mL), IL-25 (10 ng/mL), and IL-33 (5 ng/mL) (R&D Systems) to expand and maintain ILC2s for 8–10 days. Before coculture, ILC2s were rested in IL-2 (10 ng/mL) and IL-7 (10 ng/mL) for 24 hours, followed by 24 hours of IL-7 (10 ng/mL), as previously described.^{45 (link)} This resulted in reduced proliferation, activation (eg, IL-5 and IL-13 production), and smaller cell morphology while maintaining >95% viability. Cells were washed 3 times in complete RPMI 1640 media to remove cytokines before coculture experiments with eosinophils. More details are available in Fig E2, in Fig E3, A, and in the Methods in the Online Repository at www.jacionline.org.

LeSuer W.E., Kienzl M., Ochkur S.I., Schicho R., Doyle A.D., Wright B.L., Rank M.A., Krupnick A.S., Kita H, & Jacobsen E.A. (2023). Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice. The Journal of allergy and clinical immunology, 152(2), 469-485.e10.

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8

Investigating Immune Responses in Helminth Infection

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Third-stage larvae (L3) of N. brasiliensis were purified with a Baermann apparatus. After washing three times in PBS, living worms were counted. On day 0, 500 purified worms were injected subcutaneously in PBS. In addition, in some experiments an anti-CD4 depleting antibody (clone GK1.5, 250 μg/mouse, in house) was injected intraperitoneally (i.p.) at day 0 and day 2, 4, and 6 of N. brasiliensis infection. In some experiments, anti-ICOS antibody (500 μg/mouse, Bioxcell) and rat IgG isotype control (500 μg/mouse, Sigma-Aldrich) were injected i.p. at day 1 and day 3 of N. brasiliensis infection and analyzed on day 5 post-infection, or treated with anti-ICOS antibody or rat IgG isotype control every other day for six days and analyzed one day later. In some experiments, mice were treated with carrier-free recombinant murine IL-33 (1 μg/mouse, R&D Systems) intravenously (i.v.) and analyzed 24 h later or treated with IL-33 (500 ng/mouse) or IL-25 (250μg/mouse, R&D Systems) i.p. for three consecutive days and analyzed one day later. In the IL-33 rescue experiments, mice were injected i.p. with IL-33 (12.5 μg/kg body weight/day) (Brestoff et al., 2015 (link)) from day 1 to day 6 of N. brasiliensis infection and analyzed 24 h later.

Flamar A.L., Klose C.S., Moeller J.B., Mahlakõiv T., Bessman N.J., Zhang W., Moriyama S., Stokic-Trtica V., Rankin L.C., Putzel G.G., Rodewald H.R., He Z., Chen L., Lira S.A., Karsenty G, & Artis D. (2020). Interleukin-33 induces the enzyme tryptophan hydroxylase 1 to promote inflammatory group 2 innate lymphoid cell-mediated immunity. Immunity, 52(4), 606-619.e6.

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9

Isolation and Expansion of ILC2s from Murine Lungs

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Naive WT mice were intratracheally administered 500 ng/50 μL of IL-33 (R&D Systems) in PBS on days 0 and 2 and humanely killed on day 4 to isolate lung single cell suspensions as described above. Cells were depleted of Lin⁺ cells by magnetic bead separation according to the manufacturer’s recommendations (Miltenyi Biotec, San Diego, Calif). The lineage-depleted population was sorted by flow cytometry for ILC2s (Lin⁻CD45⁺CD90.2⁺Sca-1⁺ST2⁺) on a BD Biosciences FACS Aria III SORP Cell Sorter. The purity of sorted ILC2s was >99%. ILC2s were cultured in complete RPMI 1640 with IL-2 (10 ng/mL), IL-7 (10 ng/mL), IL-25 (10 ng/mL), and IL-33 (5 ng/mL) (R&D Systems) to expand and maintain ILC2s for 8–10 days. Before coculture, ILC2s were rested in IL-2 (10 ng/mL) and IL-7 (10 ng/mL) for 24 hours, followed by 24 hours of IL-7 (10 ng/mL), as previously described.^{45 (link)} This resulted in reduced proliferation, activation (eg, IL-5 and IL-13 production), and smaller cell morphology while maintaining >95% viability. Cells were washed 3 times in complete RPMI 1640 media to remove cytokines before coculture experiments with eosinophils. More details are available in Fig E2, in Fig E3, A, and in the Methods in the Online Repository at www.jacionline.org.

LeSuer W.E., Kienzl M., Ochkur S.I., Schicho R., Doyle A.D., Wright B.L., Rank M.A., Krupnick A.S., Kita H, & Jacobsen E.A. (2023). Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice. The Journal of allergy and clinical immunology, 152(2), 469-485.e10.

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10

Basophil Shape Change Induced by IL-33 and IL-25

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To determine if IL-33 and IL-25 had a direct effect on basophil shape change, purified basophils from allergic donors (n = 8) suspended in RPMI-C were incubated with PBS, IL-33 or IL-25 (both R&D Systems, MN, US) (0.1, 1, 10, 100 ng/mL) at 37 °C for 0, 30, 45, 60 or 180 seconds (s). To determine if IL-33 or IL-25 primed the basophil shape change response to eotaxin, purified basophils were incubated for 18 h at 37 °C in RPMI-C with PBS, IL-33 or IL-25 (all 10 ng/mL). Following incubation, cells were stimulated with/without eotaxin (R&D Systems, MN, US) (5 ng/mL) for 0, 30, 45, 60 or 180 s. Basophils were fixed with ice-cold 1 % PFA for 10 min to stop the reaction and cell shape change was measured by the parameter FSC via flow cytometry.

Salter B.M., Oliveria J.P., Nusca G., Smith S.G., Tworek D., Mitchell P.D., Watson R.M., Sehmi R, & Gauvreau G.M. (2016). IL-25 and IL-33 induce Type 2 inflammation in basophils from subjects with allergic asthma. Respiratory Research, 17, 5.

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Il 33

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160 protocols using il 33

Immunohistochemical Analysis of Wound Healing

Investigating IL-33 Signaling in HTNV-Infected HUVECs

Investigating IL-33 Modulation of Mast Cell Responses to Rhinovirus

Murine Cytokine Quantification via ELISA

Purification and Culture of Eosinophils

Purification and Culture of Eosinophils

Isolation and Expansion of ILC2s from Murine Lungs

Investigating Immune Responses in Helminth Infection

Isolation and Expansion of ILC2s from Murine Lungs

Basophil Shape Change Induced by IL-33 and IL-25

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