Fig 1: Lack of Orai1 results in impaired M2 phenotype development even with IL4 stimulus(A) Flow cytometric analysis of macrophage polarization in control siRNA (siC) and siOrai1-treated cells. BMDM cells were harvested 24 h after gene silencing and flow cytometry was performed to measure the levels of F4/80+ and CD11b+. Data shown are representative of three independent experiments with similar results.(B) Percentage of positive cells for F4/80+ and CD11b+ under the conditions in (A). Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, **p = 0.001 (Student's t test).(C) Flow cytometric analysis (CD80+ and CD86+) on macrophage upon respective siRNA treatment. Data shown are representative of three independent experiments.(D) Percentage of CD80+ and CD86+ positive cells. Bar graphs depict average ± SD for relative values; NS, non-significant (Student's t test) from two independent experiments.(E) Flow cytometric analysis of CD206+ and Arginase 1+ upon Orai1 silencing. Data shown are representative of three independent experiments with similar results.(F) Percentage of positive cells for M2 markers of (CD206+ and Arginase1+). Data shown are representative of three independent experiments. Bar graphs depict average ± SD for relative values, ***p = 0.001, NS, non-significant (Student's t test).(G) Cell proliferation assay for Oraiflfl with (for Orai1-/-) and without (CTRL) Ad.Cre virus. Data shown are representative of three independent experiments. Bar graphs depict BrdU-positive cells ±SD for positive cells, ***p = 0.001 (Student's t test), NS, non-significant. n = 3–4 independent replicates.(H) Flow cytometer analysis to measure the difference between Orai1fl/fl with and without Ad.Cre virus for M1, M2 markers. Data shown are representative of three independent experiments.(I) Percentage of positive cells for CD80 and CD206 under the conditions in (H). Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, NS, non-significant, ***p = 0.001 (Student's t test).(J) Immunoblot analysis showing the level of expression of iNOS, Arginase1, Orai1, and ß-actin of control and Orai1fl/fl mice with and without Ad.Cre virus. Data shown are representative of three independent experiments with similar results.(K) IV curves of Tg-induced currents at -80 mV in Orai1fl/fl with and without Ad.Cre virus in various cells. Bar graphs represent quantitation (6–8 recordings for each condition) of current intensity at -80 mV. *p = 0.05 (Student's t test).(L and M) Oxygen consumption rate (OCR) was analyzed in macrophages isolated from WT with and without 2APB (50 µM for 6 h).(N and O) OCR from WT and Orai1fl/fl macrophages treated with Ad5a iCre for 72 h for M0, M1, and M2 phenotypes. N = 3 performed in duplicate. Basal, maximal respiration, and proton leak analysis of macrophages isolated from WT with and without 2APB (50 µM for 6 h) WT and Orai1fl/fl macrophages treated with Ad5a iCre for 72 h. Mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s., not significant.
Fig 2: Orai1 expression is essential to development and proliferation of in vitro bone marrow-derived naive macrophage from wild-type (WT) mice (C57BL/6J)(A) Naive Mf (M0) cells from WT mice (C57BL/6J) were generated using L929 supplemented media. Analysis was performed using flow cytometer, and the levels of F4/80+ CD11b+ were used to show efficacy of cell maturation (Mf). n = 4 independent experiments with replicates.(B) Cell proliferation (using BrdU colorimetric assays) was evaluated in naive Mf (M0) from WT mice cells cultured in the presence or absence of 10 µM SKF96365 or 50 µM 2APB. Data shown are representative of three independent experiments with two replicates. Bar graphs depict average ±SD for BrdU-positive cells, ***p = 0.001 (Student's t test).(C) Representative Ca2+ trace (from 110 to 160 cells imaged from three separate experiments) showing Thapsigargin (Tg)-evoked ER Ca2+ depletion followed by extracellular Ca2+-induced [Ca2+]i entry in Mf cells pretreated with and without 50 µM SKF96365 for 6 h.(D) Bar graphs depict average ± SEM of the fluorescence ratio (340/380) from an average of 110–160 cells. *p = 0.05 (Student's t test).(E–G) Application of 2 µM Thapsigargin (Tg) in bath solution induced calcium currents at -80 mV in control and 50 µM SKF96365-treated Mf cells. Respective IV curves (F) of the currents (leak/background subtracted) and quantitation (8–10 individual recordings) of the current intensity at -80 mV are shown in (G). *p = 0.05 (Student's t test).(H) Representative trace showing Thapsigargin (Tg)-evoked Ca2+ entry in naive Mf cells pretreated with and without LPS, LPS +10 µM SKF96365 for 6 h, and LPS+50 µM of 2APB. n = 3–5 independent replicates for each experiment performed in duplicate.(I) Bar graphs depict average ± SEM, *p = 0.05 (Student's t test) in individual conditions as labeled.(J) Immunoblotting analysis showing expression of TRPC1, STIM1, Orai1, and ß-actin in naive Mf from WT mice cultured in the presence or absence of LPS treatment (100 ng/mL for 6 h). n = 3 independent experiments performed in duplicate.(K and L) IV curves of SOCE currents under various conditions and quantitation (6–8 recordings for each condition) of current intensity at -80 mV is shown in L. *p = 0.05.(M) Representative traces showing calcium entry in control and Orai1-silenced (siOrai1) naive Mf cells, with and without LPS treatment (100 ng/mL for 6 h). n = 3–5 independent replicates.(N) Average data ± SEM from 90 to 120 cells is provided as bar diagram, *p = 0.05 (Student's t test).(O) Thapsigargin (Tg) evoked Ca2+ entry in TRPC1-silenced (siC1) naive Mf cells, in the presence or absence of LPS treatment (100 ng/mL for 6 h). n = 3 to 4 separate experiments.(P) Bar diagram of the fluorescence ratio (340/380) from an average of ~100 cells in each condition. Bar graphs depict average ± SEM, NS, non-significant.(Q) Cell proliferation assay was performed in cells from WT mice treated with siControl (CTRL), siTRPC1, or siOrai1 cells. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SE for positive cells in four separate experiments, NS, non-significant, ***p = 0.001 (Student's t test).
Fig 3: Placenta M1 and M2 phenotype macrophage polarization following exposure to intrauterine inflammation and maternal dendrimer-based N-Acetyl Cysteine (DNAC) treatment. At embryonic (E) day 17, pregnant CD-1 mice underwent a mini-laparotomy in the lower abdomen for intrauterine injection of lipopolysaccharide (LPS). One hour later, dams received intraperitoneally injection of DNAC or phosphate-buffered saline (PBS). Placentas were collected at 6 hpi after LPS or PBS injection. (A) The placental macrophages were further identified by F4/80+ and CD11b + properties on placental leukocytes. (B) M2 macrophages was identified by CD163+ and side scatter sequentially on CD45+ CD11b+ F4/80+ macrophages. The ratios of total macrophages (C), M1 (D) and M2 (E) macrophages infiltrated in the placenta were statically compared between groups. PBS + PBS, n = 8; LPS + PBS, n = 8; LPS + DNAC, n = 7; PBS + DNAC, n = 6. *p < 0.05; **p < 0.01. Data were reported as Mean ± SEM.
Fig 4: Infiltrating Monocytes Contribute to Three Subsets of Macrophages during Myocarditis(A) Schematics of parabiosis mice.(B) Representative images of H&E-stained heart sections of the median CD45.1 (day 21 EAM) and CD45.2 (non-EAM) mice parabionts. Scale bars: 100 µm.(C) Comparison of total grafted CD11b+Ly6G-myeloid cell counts between parabionts.(D) Comparison of total cardiac macrophage counts between parabionts.(E) Flow cytometry plots showing (top left) percentage of CD45.2+CD11b+Ly6G-Lineage (CD3e, B220, NKp46, CD90.2, and Ter119)- grafted cells infiltrating the CD45.1 EAM hearts; (top middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; (top right) percentage of grafted MDM subsets; (bottom left) percentage of CD45.1+CD11b+Ly6G-Lineage grafted cells infiltrating the CD45.2 non-EAM hearts; (bottom middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; and (bottom right) percentage of grafted MDM subsets.(F) Percentages of grafted MDMs out of total number of grafted CD11b+Ly6G-Lineage- myeloid cells.(G) Percentages of grafted Ly6Chi and Ly6Clo cells out of total number of grafted F4/80-CD64+ monocytes.(H) Comparison of grafted MDM subsets defined by CCR2 and MHCII expressions between parabionts. Data are representative of two independent experiments with biological triplicates. n = 3.(C, D, F, G) Groups were compared using Student’s t test. *p < 0.05. All data were presented as mean ± SD.See also Figure S1.
Fig 5: IL-17A Trans-Signaling through Cardiac Fibroblasts Downregulates MerTK expression on Monocytes and MDMs(A and B) Representative EM images showing (A) apoptotic cells or apoptotic cellular debris internalized by Ly6Chi MDMs (arrowheads) and (B) engulfed cellular debris were largely absent in Ly6Clo MDMs. Scale bars: 2 µm.(C) Macrophage phagocytic index was calculated using the following formula: (number of engulfed apoptotic cells/total number of macrophages) × (number of macrophages with engulfed apoptotic cells/total number of macrophages) × 100.(D–G) Hearts from day 21 EAM mice.(D) Frequencies of F4/80hiCD64+ macrophages out of viable CD45+Ly6G–CD11b+ cells were assessed by flow cytometry.(E and F) MerTK MFI of F4/80hiCD64+ macrophages (E) and F4/80–CD64+ monocytes (F) in the hearts.(G) ELISA showing soluble Mer (sMer) in WT and IL-17Ra-/- EAM mice sera.(H and I) Cardiac fibroblasts were harvested from WT naive mice, whereas monocytes were sorted from EAM IL-17Ra-/- mice.(H) MerTK MFI of Ly6Chi or Ly6Clo monocytes and MDMs in vitro after 160 h post-co-culture with cardiac fibroblasts stimulated with or without IL-17A.(I) sMer detected in supernatants of the monocyte-fibroblast co-culture by ELISA.(J) Flow cytometric analysis of the frequencies of FITC+F4/80hiCD64+ macrophages in the myocardium of WT, IL-17Ra-/-, and non-treated controls.(K) Percentages of FITC+F4/80hiCD64+ macrophages out of viable CD45+Ly6G–CD11b+ cells.(L) MerTK MFI in patients with either myocarditis or ischemic cardiomyopathy.(D–G) Data are representative of five independent experiments. n = 8 – 9. (H and I) Data are representative of three independent experiments with technical triplicates. n = 3. (J and K) Data are representative of two independent experiments. n = 3. (C–G, I, K, and L) Groups were compared using Student’s t test. *p < 0.05; **p < 0.01. (H) Groups were compared using one-way ANOVA followed by Dunnett test. **p < 0.01; ****p < 0.0001. All data were presented as mean ± SD.See also Figure S7 and Tables S2 and S3.
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