Journal: Scientific Reports

Article Title: Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

doi: 10.1038/s41598-021-94843-9

Figure Lengend Snippet: Airspace eosinophilia in the developing lungs of mice. Cell counts in BALF from mice at the age of postnatal day (PND) 7, 10, 15, and 42 (n = 5–10 per group). Differential cell counts ( A ) and relative percentages of constituent cell types ( B ) are shown as stacked bar graphs [macrophages (red), neutrophils (blue), eosinophils (green), and lymphocytes (black)]. Representative images of lung sections that were immunohistochemically stained for IL-33 (Red arrows; IL-33-stained cells, Black arrows; unstained cells) ( C ). Image J quantification of IL-33 immunostained sections (n = 5–6 per group) ( D ). Cytokine levels (pg/ml) of IL-5 ( E ) and Eotaxin ( F ) in cell-free BALF of mice at PND 7, 10, 15, and 42 (n = 5 per group). Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using ANOVA followed by Tukey’s multiple comparison post hoc test. The lower limits of detection (indicated by red dotted lines) for IL-5 and Eotaxin were 0.25 pg/ml and 2.51 pg/ml, respectively. The results represent cumulative data on mice generated in two to three different litters.

Article Snippet: Sections were blocked with the blocking buffer for 20 min followed by primary antibody incubation with goat polyclonal to IL-33 (AB3626; R&D systems, Minneapolis, MN) at room temperature (RT) for 2 h. Sections were washed and incubated with diluted biotinylated secondary antibodies for 1 h at RT.

Techniques: Staining, Comparison, Generated

Journal: Scientific Reports

Article Title: Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

doi: 10.1038/s41598-021-94843-9

Figure Lengend Snippet: Airspace eosinophilia is regulated by the innate lymphoid system. Cell counts in BALF from 10-day-old wildtype (WT), IL-33 −/− , IL2rγ −/− , Rag1 −/− , and IL4Rα −/− neonates (n = 5–14 per group). Differential cell counts ( A ) and relative percentages of constituent cell types ( B ) are shown as stacked bar graphs [macrophages (red), neutrophils (blue), eosinophils (green), and lymphocytes (black)]. Cytokine levels (pg/ml) of IL-5 (C) and Eotaxin ( D ) in cell-free BALF from 10-day-old wildtype, IL-33 −/− , IL2rγ −/− , Rag1 −/− , and IL4Rα −/− neonates (n = 5 per group). The lower limits of detection (indicated by red dotted lines) for IL-5 and Eotaxin were 0.25 pg/ml and 2.51 pg/ml, respectively. Error bars represent SEM. ** p < 0.01, *** p < 0.001 using ANOVA followed by Tukey’s multiple comparison post hoc test. ( E ) Gating Strategy and representative scatter plots for flow cytometric characterization of ILC2’s (CD45 + Lin - CD278 + CD90.2 + ST2 + ) in whole-lung single-cell suspension from IL-33-treated IL4Rα −/− mice (n = 3 per group). ( F ) Percentage of ILC2s in whole-lung single-cell suspension from IL-33-treated IL2rγ −/− , saline-treated IL4Rα −/− , IL-33-treated IL4Rα −/− , and IL-33-treated IL4Rα +/- mice. Error bars represent SEM. ** p < 0.01, using ANOVA followed by Tukey’s multiple comparison post hoc test. The results represent cumulative data from mice generated in three different litters.

Article Snippet: Sections were blocked with the blocking buffer for 20 min followed by primary antibody incubation with goat polyclonal to IL-33 (AB3626; R&D systems, Minneapolis, MN) at room temperature (RT) for 2 h. Sections were washed and incubated with diluted biotinylated secondary antibodies for 1 h at RT.

Techniques: Comparison, Suspension, Saline, Generated

Journal: Scientific Reports

Article Title: Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

doi: 10.1038/s41598-021-94843-9

Figure Lengend Snippet: Levels of ligands for IL4Rα receptor in airspaces. ( A ) Cytokine levels (pg/ml) of IL-13 in BALF from WT mice at the age of postnatal day (PND) 7, 10, 15, and 42 (n = 5 per group). ( B ) Cytokine levels (pg/ml) of IL-13 in BALF from 10-day-old wildtype (WT), IL-33 −/− , IL2rγ −/− , Rag1 −/− , and IL4Rα −/− neonates (n = 5 per group). (C) Cytokine levels (pg/ml) of IL-4 in BALF from WT mice at PND 7, 10, 15, and 42 (n = 5 per group). (D) Cytokine levels (pg/ml) of IL-4 in BALF from 10-day-old WT, IL-33 −/− , IL2rγ −/− , Rag1 −/− , and IL4Rα −/− neonates (n = 5 per group). Error bars represent SEM. *p < 0.05, **p < 0.01, using ANOVA followed by Tukey’s multiple comparison post hoc test. The lower limit of detection (indicated by red dotted lines) for IL-4 and IL-13 were 0.03 pg/ml and 0.08 pg/ml, respectively. The results represent cumulative data from mice generated in three different litters.

Article Snippet: Sections were blocked with the blocking buffer for 20 min followed by primary antibody incubation with goat polyclonal to IL-33 (AB3626; R&D systems, Minneapolis, MN) at room temperature (RT) for 2 h. Sections were washed and incubated with diluted biotinylated secondary antibodies for 1 h at RT.

Techniques: Comparison, Generated

Journal: Scientific Reports

Article Title: Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

doi: 10.1038/s41598-021-94843-9

Figure Lengend Snippet: Myeloid-IL4Rα is essential for IL-13 and IL-5/eotaxin-induced lung eosinophilia and eosinophil chemoattractants are significantly reduced in the BALF of IL-13 treated mye-IL4Rα −/− versus IL-13 treated WT mice. Scheme for IL-13-induced lung eosinophilia ( A ). Total number of eosinophils recovered from IL-13 treated WT, IL4Rα −/− and mye-IL4Rα −/− mice (n = 4 per group) ( B ). Representative photomicrograph of BALF cytospins from IL-13-treated WT ( C ), IL4Rα −/− ( D ), and mye-IL4Rα −/− ( E ) mice (red arrows, eosinophils; black arrows, macrophages). Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using ANOVA followed by Tukey’s multiple comparison post hoc test. Cytokine levels (pg/ml) of CCL3 (MIP1α) ( F ), CCL4 (MIP1β) ( G ), CCL5 (Rantes) ( H ), CCL7 ( I ), CCL11 (Eotaxin 1) ( J ), CCL12 ( K ), CCL17 ( L ), CCL22 ( M ), CCL24 (Eotaxin 2) ( N ) , and IL-33 ( O ) in cell-free BALF from saline- or IL-13-treated WT and mye-IL4Rα −/− adult mice. The lower limit of detection for CCL3 (MIP1α), CCL4 (MIP1β), CCL5 (Rantes), CCL7, CCL11 (Eotaxin 1), CCL12, CCL17, CCL22, and CCL24 (Eotaxin 2) were 0.23 pg/ml, 4.0 pg/ml, 4.0 pg/ml, 0.27 pg/ml, 0.42 pg/ml, 3.41 pg/ml, 0.22 pg/ml, 5.03 pg/ml, 0.42 pg/ml, 0.60 pg/ml, and 4.06 pg/ml, respectively. The lower limit of detection for CCL5 (Rantes) and CCL11 (Eotaxin 1) are indicated by red dotted lines. Error bars represent SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using 2-way ANOVA followed by Tukey's multiple comparison post hoc test. n = 3–5. Results in panels B-E are representative of three independent experiments. Data in panels F-O were generated using BALF from 3 to 5 independent animals.

Article Snippet: Sections were blocked with the blocking buffer for 20 min followed by primary antibody incubation with goat polyclonal to IL-33 (AB3626; R&D systems, Minneapolis, MN) at room temperature (RT) for 2 h. Sections were washed and incubated with diluted biotinylated secondary antibodies for 1 h at RT.

Techniques: Comparison, Saline, Generated

Journal: Scientific Reports

Article Title: Myeloid-IL4Rα is an indispensable link in IL-33-ILCs-IL-13-IL4Rα axis of eosinophil recruitment in murine lungs

doi: 10.1038/s41598-021-94843-9

Figure Lengend Snippet: IL4Rα deletion in myeloid cells does not compromise responsiveness of eosinophils to IL-5/eotaxin-mediated recruitment into the airspaces. Total number of eosinophils recovered in the femoral bone marrow ( A : bone marrow cytospins, red arrows depict eosinophils; B : eosinophil counts) and blood ( C : blood cytospins; red arrows depict eosinophils; D : eosinophil counts) from WT and mye-IL4Rα −/− mice (n = 4 per group). Scheme for IL-5/CCL11/CCL24-induced lung eosinophilia ( E ). Total number of eosinophils recovered from IL-5/CCL11/CCL24-treated WT and mye-IL4Rα −/− mice (n = 4 per group) ( F ). Error bars represent SEM. Student’s t-test. ns; non-significant. ( G ) A conceptual overview of the cellular and molecular players during eosinophil recruitment. Release of IL-33 from the alveolar epithelial cells induces the development of ILC2s. ILC2-derived IL-13 binds to IL4Rα on myeloid cells and the latter produces detectable levels of IL-13 and IL-4. On the downstream, eosinophil chemoattractants are produced by myeloid cells, or indirectly by non-myeloid cells, that in turn, recruit eosinophils into the airspaces. Data in panels A-F were generated using BALF from 3 to 5 independent animals.

Article Snippet: Sections were blocked with the blocking buffer for 20 min followed by primary antibody incubation with goat polyclonal to IL-33 (AB3626; R&D systems, Minneapolis, MN) at room temperature (RT) for 2 h. Sections were washed and incubated with diluted biotinylated secondary antibodies for 1 h at RT.

Techniques: Derivative Assay, Produced, Generated

Journal: Gut

Article Title: A signaling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

doi: 10.1136/gutjnl-2016-312779

Figure Lengend Snippet: A. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin, chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in untreated wild-type and L635-treated wild-type mice. B. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in untreated IL33KO and L635-treated IL33KO mice. C. Quantification of Mist1 positive nuclei per 20× field. L635-treated IL33KO mice had significantly fewer Mist1-positive cells compared to untreated IL33KO mice, and significantly more Mist1-positive cells compared wild-type L635-treated mice. D. Quantification of SPEM cells determined by percent of GIF positive cells co-positive for CD44v9 and GSII-lectin. L635-treated IL33KO mice have significantly fewer SPEM cells compared to wild-type L635-treated mice. E. Quantification of proliferative SPEM cells determined by the percent of SPEM cells positive for Ki67. L635-treated IL33KO mice have significantly fewer proliferative SPEM cells compared to wild-type L635-treated mice. F. Quantification of F4/80-positive macrophages (red) and F4/80 and CD163 co-positive M2 macrophages (green) per 20× field. L635-treated IL33KO mice had similar F4/80-expressing macrophage infiltration as wild-type L635-treated mice, but significantly fewer F4/80 macrophages were co-positive for CD163. * denotes significant difference (p<0.05) compared to untreated mice, ✚ denotes significant difference (p<0.05) compared to L635-treated wild-type mice. White dashed boxes are magnified in the insets. Scale bars = 100 μm.

Article Snippet: The following primary antibodies were incubated overnight at 4°C in Antibody Diluent with Background Reducing Components (DakoCytomation): goat anti-intrinsic factor (1:1000, a gift from Dr. David Alpers, Washington University, St. Louis, MO), rat anti-Ki67 (1:50, BioLegend clone 16A8), rabbit anti-Ki67 (1:500, Cell Signaling), rat anti-F4/80 (1:500; Invitrogen, Grand Island, NY), rabbit anti-Mist1 (1:1000, a gift from Jason Mills, Washington University, St. Louis, MO), rabbit anti-IL-13rα (1:250, Abcam, 1:500), rat anti-Cd44v9 (1:25,000; Cosmo Bio, Japan), mouse anti-CD163 (1:200, NeoMarkers, Fremont, CA and goat anti-IL-33 (1:200; R&D Systems).

Techniques: Immunofluorescence, Staining, Marker, Variant Assay, Expressing

Journal: Gut

Article Title: A signaling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

doi: 10.1136/gutjnl-2016-312779

Figure Lengend Snippet: A. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin, chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in untreated ST2 (IL33 receptor) knockout mice and L635-treated ST2 knockout mice. B. Quantification of Mist1 positive nuclei per 20× field. L635-treated ST2KO mice had significantly fewer Mist1-positive cells compared to untreated ST2KO mice, although some Mist1-positive cells remained. C. Quantification of SPEM cells determined by percent of GIF positive cells co-positive for CD44v9 and GSII-lectin. L635-treated ST2KO mice did not have significant changes in SPEM cell number compared to untreated ST2KO mice. D. Quantification of proliferative SPEM cells determined by the percent of SPEM cells positive for Ki67. Although L635-treated ST2KO had very few SPEM cells, some of the SPEM cells were proliferating so there is a significantly higher percent of proliferative SPEM cells in L635-treated ST2KO mice compared to untreated ST2KO mice. E. Quantification of F4/80-positive macrophages (red) and F4/80 and CD163 co-positive M2 macrophages (green) per 20× field. L635-treated ST2KO mice have significant F4/80-positive macrophage infiltration compared to untreated ST2KO mice. L635-treated ST2KO mice also have significantly more F4/80 and CD163 co-positive cells compared to untreated ST2KO mice, however the percent of F4/80 macrophages that are co-positive for CD163 is around 10% in both mouse models. * denotes significant difference (p<0.05) compared to untreated mice. White dashed boxes are magnified in the insets. Scale bars = 100 μm.

Article Snippet: The following primary antibodies were incubated overnight at 4°C in Antibody Diluent with Background Reducing Components (DakoCytomation): goat anti-intrinsic factor (1:1000, a gift from Dr. David Alpers, Washington University, St. Louis, MO), rat anti-Ki67 (1:50, BioLegend clone 16A8), rabbit anti-Ki67 (1:500, Cell Signaling), rat anti-F4/80 (1:500; Invitrogen, Grand Island, NY), rabbit anti-Mist1 (1:1000, a gift from Jason Mills, Washington University, St. Louis, MO), rabbit anti-IL-13rα (1:250, Abcam, 1:500), rat anti-Cd44v9 (1:25,000; Cosmo Bio, Japan), mouse anti-CD163 (1:200, NeoMarkers, Fremont, CA and goat anti-IL-33 (1:200; R&D Systems).

Techniques: Immunofluorescence, Staining, Marker, Variant Assay, Knock-Out

Journal: Gut

Article Title: A signaling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

doi: 10.1136/gutjnl-2016-312779

Figure Lengend Snippet: A. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin, chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in wild-type mice treated with non-specific IgG or the following L635 treated mice: non-specific IgG, anti-IL-5 and anti-IL-9. B. Quantification of Mist1 positive nuclei per 20× field. IL-5 or IL-9 depleted L635-treated mice had significant loss of Mis1-positive cells similar to non-specific IgG L635-treated mice. C. Quantification of SPEM cells determined by percent of GIF positive cells co-positive for CD44v9 and GSII-lectin. IL-5 or IL-9 depleted L635-treated mice have a significant percent of SPEM cells similar to non-specific IgG L635-treated mice. D. Quantification of proliferative SPEM cells determined by the percent of SPEM cells positive for Ki67. IL-5 or IL-9 depleted mice have a significant percent of proliferating SPEM cells similar to non-specific IgG L635-treated mice. E. Quantification of F4/80-positive macrophages (red) and F4/80 and CD163 co-positive M2 macrophages (green) per 20× field. IL-5 or IL-9 depleted mice had significant infiltration of F4/80-positive macrophages, most of which were co-positive for CD163 similar to non-specific IgG L635-treated mice. * denotes significant difference (p<0.05) compared to non-specific IgG-treated mice without L635-treatment. White dashed boxes are magnified in the insets. Scale bars = 100 μm.

Article Snippet: The following primary antibodies were incubated overnight at 4°C in Antibody Diluent with Background Reducing Components (DakoCytomation): goat anti-intrinsic factor (1:1000, a gift from Dr. David Alpers, Washington University, St. Louis, MO), rat anti-Ki67 (1:50, BioLegend clone 16A8), rabbit anti-Ki67 (1:500, Cell Signaling), rat anti-F4/80 (1:500; Invitrogen, Grand Island, NY), rabbit anti-Mist1 (1:1000, a gift from Jason Mills, Washington University, St. Louis, MO), rabbit anti-IL-13rα (1:250, Abcam, 1:500), rat anti-Cd44v9 (1:25,000; Cosmo Bio, Japan), mouse anti-CD163 (1:200, NeoMarkers, Fremont, CA and goat anti-IL-33 (1:200; R&D Systems).

Techniques: Immunofluorescence, Staining, Marker, Variant Assay

Journal: Gut

Article Title: A signaling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

doi: 10.1136/gutjnl-2016-312779

Figure Lengend Snippet: A. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin, chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in untreated IL13KO mice and L635-treated IL13KO mice. B. Quantification of Mist1 positive nuclei per 20× field. L635-treated IL13KO mice had significantly fewer Mist1-positive cells compared to untreated IL13KO mice, although some Mist1-positive cells remained. C. Quantification of SPEM cells determined by percent of GIF positive cells co-positive for CD44v9 and GSII-lectin. L635-treated IL13KO mice have a significantly higher percent of SPEM cells, however this number is still relatively low. D. Quantification of proliferative SPEM cells determined by the percent of SPEM cells positive for Ki67. L635-treated IL13KO had a few SPEM cells and some of the SPEM cells were proliferating so there is a significantly higher percent of proliferative SPEM cells in L635-treated IL13KO mice compared to untreated IL13KO mice. E. Quantification of F4/80-positive macrophages (red) and F4/80 and CD163 co-positive M2 macrophages (green) per 20× field. L635-treated IL13KO mice have significant F4/80-positive macrophage infiltration compared to untreated IL13KO mice. L635-treated IL13KO mice also have significantly more F4/80 and CD163 co-positive cells compared to untreated IL13KO mice, however the percent of F4/80 macrophages that are co-positive for CD163 is around 15% in both mouse models. * denotes significant difference (p<0.05) compared to untreated mice. White dashed boxes are magnified in the insets. Scale bars = 100 μm.

Article Snippet: The following primary antibodies were incubated overnight at 4°C in Antibody Diluent with Background Reducing Components (DakoCytomation): goat anti-intrinsic factor (1:1000, a gift from Dr. David Alpers, Washington University, St. Louis, MO), rat anti-Ki67 (1:50, BioLegend clone 16A8), rabbit anti-Ki67 (1:500, Cell Signaling), rat anti-F4/80 (1:500; Invitrogen, Grand Island, NY), rabbit anti-Mist1 (1:1000, a gift from Jason Mills, Washington University, St. Louis, MO), rabbit anti-IL-13rα (1:250, Abcam, 1:500), rat anti-Cd44v9 (1:25,000; Cosmo Bio, Japan), mouse anti-CD163 (1:200, NeoMarkers, Fremont, CA and goat anti-IL-33 (1:200; R&D Systems).

Techniques: Immunofluorescence, Staining, Marker, Variant Assay

Journal: Gut

Article Title: A signaling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

doi: 10.1136/gutjnl-2016-312779

Figure Lengend Snippet: A. Immunofluorescence staining for chief cell transcription factor Mist1, mucus marker GSII-lectin, chief cell granule marker gastric intrinsic factor (GIF), SPEM marker CD44 variant isoform 9 (CD44v9), proliferation marker Ki67, macrophage marker F4/80, M2 macrophage marker CD163 and nuclei marker DAPI in ST2KO mice treated with IL-13 (ST2KOIL13), ST2KO mice treated with a non-specific IgG and L635 (ST2KOL635), or ST2KO mice treated with IL-13 and L635 (ST2KOL635-IL13). B. Quantification of Mist1 positive nuclei per 20× field. ST2KOL635-IL13 mice had significant loss of Mist1-positive cells similar to ST2KOL635. C. Quantification of SPEM cells determined by percent of GIF positive cells co-positive for CD44v9 and GSII-lectin. ST2KOL635-IL13 mice have a significantly higher percent of SPEM cells compared to ST2KOIl13 and ST2KOL635 mice. D. Quantification of proliferative SPEM cells determined by the percent of SPEM cells positive for Ki67. Although there is more SPEM in ST2KOL635-IL13 mice, there is no significant difference in proliferative SPEM. E. Quantification of F4/80-positive macrophages (red) and F4/80 and CD163 co-positive M2 macrophages (green) per 20× field. ST2KOL635-IL13 mice exhibit similar levels of F4/80-positive macrophage infiltration to ST2KOL635 mice, but there are significantly more F4/80 and C163 co-positive M2 macrophages in ST2KOL635-IL13 mice. * denotes significant difference (p<0.05) compared to untreated mice, ✚ denotes significant difference (p<0.05) compared to ST2KO non-specific IgG-treated L635-treated mice. White dashed boxes are magnified in the insets. Scale bars = 100 μm.

Article Snippet: The following primary antibodies were incubated overnight at 4°C in Antibody Diluent with Background Reducing Components (DakoCytomation): goat anti-intrinsic factor (1:1000, a gift from Dr. David Alpers, Washington University, St. Louis, MO), rat anti-Ki67 (1:50, BioLegend clone 16A8), rabbit anti-Ki67 (1:500, Cell Signaling), rat anti-F4/80 (1:500; Invitrogen, Grand Island, NY), rabbit anti-Mist1 (1:1000, a gift from Jason Mills, Washington University, St. Louis, MO), rabbit anti-IL-13rα (1:250, Abcam, 1:500), rat anti-Cd44v9 (1:25,000; Cosmo Bio, Japan), mouse anti-CD163 (1:200, NeoMarkers, Fremont, CA and goat anti-IL-33 (1:200; R&D Systems).

Techniques: Immunofluorescence, Staining, Marker, Variant Assay

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Nuclear localization of the IL-33 and reduced with the onset of labor. The sections fixed paraffin-embedded of human myometrium were immune-stained with anti-IL-33 antibody. (A) Immunohistochemistry results showed that whether in TNL or PNL tissue, IL-33 mostly located in the nuclear while reduced sharply and emerged in the cytoplasm with the initiation of labor as shown in figure TL and PTL. Quantification of IL-33 expression within the nucleus region showed apparent differences between labor and non-labor tissue (n=4). (B) The sections of human myometrium were visualized by an Alexa Flour 594 secondary antibody labeled with IL-33 (Red), and nuclei were stained with DAPI (Blue). Analysis of nuclei was performed by confocal microscopy, fluorescence signals of IL-33 and nuclei were superimposed. Immunofluorescence staining results reflected the same phenomenon. (C) From Western blots, we also can see that non-labor groups had more nuclear expression and less cytoplasmic expression of IL-33 compared to labor groups while the total IL-33 had no obvious differences between groups. QT-PCR discovered no apparent alteration in the levels of IL-33 mRNA. Each value represents the mean ± standard deviation (SD) of three independent experiments. ** P<0.01, *** P<0.001, bar 50 μm.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Staining, Immunohistochemistry, Expressing, Labeling, Confocal Microscopy, Fluorescence, Immunofluorescence, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Dynamic changes of IL-33 position during LPS stimulation. (A) Localization analysis of confocal laser scanning microscopy images of IL-33 with the stimulation of 10 μg/ml LPS, we could see that IL-33 in the nucleus declined sharply compared with the control group, especially in 3 hours. However, with the longer time of the LPS treatment, the expression of IL-33 commenced to rise again from 6 hours and reached the peak at 12 hours. The lower panel is the percentage of cells of which IL-33 expressed mainly in the nucleus (n=3). (B) After the primary cells were loaded with LPS for different times, cytoplasmic and nuclear proteins were isolated. In the cytoplasmic and nucleus fraction IL-33 were quantified by Western blotting, the experiment was performed as described for . Each experiment was performed at least three times while shown are representative results (n=3). From the blots, we known that whether cytoplasmic or nucleus fraction IL-33 levels presented the same change trend as immunofluorescence. * P<0.05, ** P<0.01, bar 50 μm.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Confocal Laser Scanning Microscopy, Expressing, Isolation, Western Blot, Immunofluorescence

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing enhanced LPS-induced expression of calcium channels and the intracellular calcium concentration. Primary myometrium cells were transfected with IL-33 or non-targeting (as control) siRNAs and treated with 10 μg/ml LPS or PBS (as control) for 6 hours. (A) Immunofluorescence staining analysis performed with fluo-3AM (green), nuclei were stained with DAPI (blue) (n=3). (B) Western blot analysis for expression levels of Cav 3.1 and Cav 3.2 in stably transfected and non-transfected myometrium cells with treatment as indicated (n=5). * P<0.05, ** P <0.01, bar 50 μm.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Expressing, Concentration Assay, Transfection, Immunofluorescence, Staining, Western Blot, Stable Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing highlighted LPS-induced endoplasmic reticulum stress response. Based on the discovery that calcium ions affected endoplasmic reticulum stress, Western blot and QT-PCR were we further to explore the expression of endoplasmic reticulum stress in tissues from protein and mRNA level. (A) The protein levels of P-IRE1α and XBP1s in the TL and PTL groups were higher than those in the TNL and PNL groups while there was no alteration in the level of GRP78 protein(n=6). (B) The mRNA level of XBP1s in the PTL groups was higher than that in the PNL groups (n=6). Furthermore, in order to illustrate the protein level changes of endoplasmic reticulum stress during labor, LPS was used to stimulate primary uterine smooth muscle cells for different time course and then Western blot was used to detect the alteration of endoplasmic reticulum stress. (C) It was found that the protein level of pIRE1α reached its peak at 10 minutes while XBP1s at 15 minutes, while the protein level of GRP78 did not change significantly (n=5). We also detected apparent alteration in endoplasmic reticulum stress protein during cells stimulated based knockdown experiments targeting IL-33. (D) It revealed that the endoplasmic reticulum stress response in the siRNA-based group was more obvious compared with the LPS stimulated directly especially at 30 minutes (n=5). * P<0.05, ** P<0.01, *** P<0.005, ****P<0.001

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Western Blot, Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and ER stress response affected COX-2 expression in myometrium cells. (A) In the process of studying whether IL-33 affects COX-2, we found that the COX-2 expression in the siRNA-mediated group was significantly increased compared with the LPS alone group (n=5). (B) Western blot analyses showing protein expression of COX-2 in myometrium cells was decreased following treatment with LPS for 12 hours (n=5). * P<0.05, ** P<0.01. Each value represents the mean ± standard deviation (SD) of three independent experiments.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 knockdown enhanced LPS-induced NF-κB and p38/MAPK signaling pathways. (A) Relative levels of p-P38, P38, p-NF-κB and NF-κB were assessed by western blot analysis at the indicated time point after LPS (10 μg/ml) stimulation. Phosphorylation levels of P38 and NF-κB increased gradually with LPS stimulation and peaked at 15 minutes and 1 hour, respectively(n=5). (B) Western blot analysis of p-P38, P38, p-NF-κB and NF-κB expression in cells transfected with siRNA targeting IL-33 after treatment with LPS for 30minutes. Compared with the LPS group, the protein expression of phosphorylated P38 and NF-κB were increased in the LPS + siRNA IL-33 group(n=5). (C) The protein level of COX-2 was decreased when SB-202190 and JSH-23 blocked p38/MAPK and NF-KB signaling pathway, respectively(n=5). * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Western Blot, Expressing, Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and cytoplasmic calcium influence the expression of IL-8 and IL-6. Compared with the LPS group, the expression of IL-8 and IL-6 was increased in the LPS+IL-33 siRNA group and decreased in the LPS+BAPTA-AM group (n=5). * P <0.05.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Model for the role of IL-33 in the myometrium participates in maintaining a uterine quiescent state at the tissue-to-cellular level during late pregnancy.

Article Snippet: Primary antibody incubation was performed overnight at 4°C with goat monoclonal anti-IL-33 (1:200, MAB36253, R&D) diluted in 0.2% BSA in PBS.

Techniques: