murine neutrophils  (R&D Systems)

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: FXR expression is markedly decreased in neutrophils from patients with active IBD. (A) Expression of FXR (encoded by NR1H4 ) in the colonic biopsies from healthy controls (HC, n = 20), patients with active CD (A‐CD, n = 20), patients with CD in remission (R‐CD, n = 20), patients with active UC (A‐UC, n = 20), and patients with UC in remission (R‐UC, n = 20) was analyzed by qRT‐PCR. (B) CD19 + B cells, CD56 + NK cells, CD4 + T cells, CD8 + T cells, CD14 + monocytes, and CD66b + neutrophils were isolated from peripheral blood of healthy donors ( n = 6) using immunomagnetic microbeads and FXR expression was determined by qRT‐PCR. (C) Expression of FXR in peripheral blood neutrophils from HC ( n = 30), A‐CD ( n = 30), R‐CD ( n = 30), A‐UC ( n = 30), and R‐UC ( n = 30) was analyzed by qRT‐PCR. (D) FXR expression in peripheral blood neutrophils from six HC, six A‐CD, six R‐CD, six A‐UC, and six R‐UC individuals was analyzed by flow cytometry. Quantitative analysis and representative histogram plot showed mean fluorescence intensity (MFI) of FXR expression. (E) Neutrophils (3 × 10 6 /mL) from healthy donors ( n = 6) were stimulated with lipopolysaccharide (LPS, 100 ng/mL), Flagellin (100 ng/mL), peptidoglycan (PGN, 1 µg/mL), Pam3CSK4 (1 µg/mL), or fibroblast‐stimulating lipopeptide‐1 (FSL‐1, 100 ng/mL) for 3 h. The expression of FXR was determined by qRT‐PCR. (F–G) Correlations between FXR mRNA levels in peripheral blood neutrophils and disease activity in patients with CD (F, n = 60; CDAI: Crohn's disease activity index; SES‐CD: simple endoscopic score for Crohn's disease) or patients with UC (G, n = 60; Mayo index; UCEIS, ulcerative colitis endoscopic index of severity). Data were expressed as mean ± SEM. Tukey's test (A–E) and Spearman's correlation analysis (F and G) were used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Fluorescence, Activity Assay

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: FXR‐deficient mice develop more severe colitis under DSS induction or Citrobacter rodentium infection. (A–G) Eight‐week‐old WT and Fxr −/− mice were administered 2% DSS in drinking water for 7 days, followed by another 3 days of regular water consumption to induce colitis ( n = 5/group). All mice were sacrificed on Day 10. (B) Changes in body weight during the disease course. (C) Representative image and quantification of colon length on Day 10. (D) Expression of inflammatory mediators in the colonic tissues was measured by qRT‐PCR. (E) Hematoxylin and eosin (H&E) staining and histopathological scores were applied to assess the severity of colitis. Scale bars, 200 µm. (F) Flow cytometric analysis of lamina propria‐infiltrating neutrophils. (G) Representative immunofluorescence staining of specific markers for neutrophil extracellular traps (NETs) in the colonic sections, with quantification of NET‐forming neutrophils. Scale bars, 50 µm. (H–N) Eight‐week‐old WT and Fxr −/− mice were orally administered with Citrobacter rodentium ( C. rodentium , 2 × 10 9 CFU/mouse) after fasting for 10 h on Day 0 to induce colitis ( n = 5/group). All mice were sacrificed on Day 8. (I) Changes in body weight over the 8‐day modeling period. (J) The gross morphology and length of colon on Day 8. (K) Expression of inflammatory mediators in the colonic tissues was analyzed by qRT‐PCR. (L) H&E staining and histopathological scores were applied to assess the severity of colitis. Scale bars, 200 µm. (M) Flow cytometric analysis of lamina propria‐infiltrating neutrophils. (N) Immunofluorescence staining of NET markers in the colonic sections, with quantification of NET‐forming neutrophils. Scale bars, 50 µm. Data were expressed as mean ± SEM. Tukey's test (B, C, E, F, and G) and Student's t ‐test (D and I–N) were used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Infection, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: Adoptive transfer of FXR‐deficient neutrophils exacerbates DSS‐induced colitis in mice. (A) Purified bone marrow (BM)‐derived neutrophils isolated from WT or Fxr −/− donors were intravenously transferred (2 × 10 6 /mouse) on Days 0, 3, 5, and 7 ( n = 5/group). All recipients were administered 2% DSS in drinking water for 7 days, followed by another 3 days of regular water consumption to induce colitis. All mice were sacrificed on Day 10. (B) Changes in body weight over a 10‐day modeling period. (C) The gross morphology and length of colon on Day 10. (D) Expression of inflammatory mediators in the colonic tissues was analyzed by qRT‐PCR. (E) H&E staining and histopathological scores were applied to assess the severity of colitis. Scale bars, 200 µm. (F) Flow cytometric analysis of lamina propria‐infiltrating neutrophils. (G) Representative immunofluorescence staining of NET markers in the colonic sections, with quantification of NET‐forming neutrophils. Scale bars, 30 µm. Data were expressed as mean ± SEM. Student's t ‐test was used for statistical analysis. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Adoptive Transfer Assay, Purification, Derivative Assay, Isolation, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: FXR‐deficient neutrophils exhibit augmented proinflammatory activity. BM‐derived neutrophils ( n = 3/group) were harvested from WT and Fxr −/− mice for RNA sequencing analysis. (A) Gene set enrichment analysis (GSEA) of transcriptomic profiles. NES, normalized enrichment score. (B) Heatmap of genes associated with neutrophil functions. TLRs, Toll‐like receptors; ROS, reactive oxygen species. (C) Representative histogram and quantification of MFI of the ROS probe CM‐H 2 DCFDA in BM‐derived neutrophils (1 × 10 6 /mL) after 3 h stimulation with LPS (300 ng/mL) ( n = 5/group). (D) Representative co‐staining of DNA, citrullinated histone‐3 (CitH3), and myeloperoxidase (MPO) to assess NET formation in BM‐derived neutrophils (1 × 10 6 /mL) after treatment with phorbol 12‐myristate 13‐acetate (PMA, 100 ng/mL) for 3 h ( n = 5/group). Scale bars, 50 µm. Quantification of NET‐forming neutrophils is shown in the bar chart. (E) Neutrophil migration was assessed using a Transwell assay, in which BM‐derived neutrophils (1 × 10 6 /mL) were seeded in the upper chamber and stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP, 50 nM) in the lower chamber for 2 h ( n = 5/group). Migrating neutrophils were quantified per high‐power field (HPF). Scale bars, 25 µm. Data were expressed as mean ± SEM. Tukey's test was used for statistical analysis. ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Activity Assay, Derivative Assay, RNA Sequencing, Staining, Migration, Transwell Assay

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: FXR suppresses proinflammatory functions in neutrophils through the mTORC1 pathway. (A) GSEA analysis of the transcriptomic data shown in Figure . (B) Heatmap of genes involved in the mTORC1‐glycolysis pathway. (C and D) Phospho‐flow analysis of mTORC1 substrates in BM‐derived neutrophils (3 × 10 6 /mL) under 3 h of LPS (300 ng/mL) treatment ( n = 5/group). P‐mTOR, phospho‐mTOR; p‐S6, phospho‐S6; p‐4E‐BP1, phospho‐4E‐BP1. (E) Representative histogram and quantification of 2‐NBDG fluorescence intensity, a fluorescent glucose analog used to monitor glucose uptake, in BM‐derived neutrophils (3 × 10 6 /mL) after 3 h of LPS (300 ng/mL) stimulation ( n = 5/group). (F) BM‐derived neutrophils (2 × 10 6 /mL) were treated with or without LPS (300 ng/mL) for 3 h, and the extracellular acidification rate (ECAR) was measured using a Seahorse analyzer ( n = 3/group). Glu, glucose; Oli, oligomycin; 2‐DG, 2‐Deoxy‐D‐glucose. (G) BM‐derived neutrophils (1 × 10 6 /mL) were treated with LPS (300 ng/mL) in the presence or absence of rapamycin (2 µM) or 2‐DG (5 mM) for 3 h. ROS production was determined by flow cytometry using CM‐H 2 DCFDA ( n = 5/group). (H) BM‐derived neutrophils (1 × 10 6 /mL) were treated with PMA (100 ng/mL) in the presence or absence of rapamycin (2 µM) or 2‐DG (5 mM) for 3 h. NET formation was detected by immunofluorescence staining. Scale bars, 50 µm. Quantification of NET‐forming neutrophils is shown in the bar chart ( n = 5/group). (I) BM‐derived neutrophils (1 × 10 6 /mL) pretreated with or without rapamycin (2 µM) or 2‐DG (5 mM) were placed in the upper chamber of the Transwell insert, and fMLP (50 nM) was added to the lower chamber for 2 h ( n = 5/group). The migration of neutrophils was detected as described in Figure . Scale bars, 25 µm. Data were expressed as mean ± SEM. Tukey's test (E–I) and Student's t ‐test (D) were used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Immunofluorescence, Staining, Migration

Journal: MedComm

Article Title: Dysregulation of Farnesoid X Receptor on Neutrophil Homeostasis Exacerbates Intestinal Inflammation via the mTORC1‐Glycolysis Signaling Pathway

doi: 10.1002/mco2.70637

Figure Lengend Snippet: FXR signaling suppresses proinflammatory functions in neutrophils from IBD patients. (A) Peripheral blood (PB) neutrophils (3 × 10 6 /mL) were isolated from patients with active CD ( n = 3) and treated with or without INT‐747 (40 µM) for 3 h. Total RNA was extracted and subjected to RNA sequencing, followed by gene set variation analysis (GSVA) to assess transcriptomic changes. (B) Representative co‐staining of DNA, CitH3, and MPO to assess NET formation in PB neutrophils (1 × 10 6 /mL) isolated from healthy donors (HC, n = 6), patients with active CD ( n = 6) and patients with active UC ( n = 6) after 3 h of PMA (100 ng/mL) stimulation in the presence or absence of INT‐747 (40 µM). Scale bars, 50 µm. Quantification of NET‐forming neutrophils is shown in the bar chart. (C) PB neutrophils (1 × 10 6 /mL) isolated from healthy donors (HC, n = 6), patients with active CD ( n = 6), and patients with active UC ( n = 6) were pretreated with or without INT‐747 (40 µM) and seeded in the upper chamber of the Transwell insert. IL‐8 (20 ng/mL) was added to the lower chamber to induce migration for 2 h. The migrated neutrophils were quantified as described in Figure . Scale bars, 25 µm. (D–F) PB neutrophils (3 × 10 6 /mL) isolated from healthy donors, patients with active CD, and patients with active UC were stimulated with LPS (300 ng/mL) in the presence or absence of INT‐747 (40 µM) for 3 h. Phosphorylation of mTORC1 substrates was analyzed by phospho‐flow cytometry ( n = 6/group). (G) ECAR levels in neutrophils treated as in (D–F) were measured using a Seahorse analyzer ( n = 3/group). Glu, glucose; Oli, oligomycin. Data were expressed as mean ± SEM. Student's t ‐test (B and C), and Tukey's test (D–G) were used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The isolation of neutrophils from the bone marrow of mice was performed using a murine neutrophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec; Bergisch Gladbach, Germany).

Techniques: Isolation, RNA Sequencing, Staining, Migration, Phospho-proteomics, Flow Cytometry

Journal: eLife

Article Title: Secretory leukocyte protease inhibitor influences periarticular joint inflammation in Borrelia burgdorferi -infected mice

doi: 10.7554/eLife.104913

Figure Lengend Snippet: ( A, B ) Infiltrating cell population analysis of tibiotarsal joint tissues of infected WT and Slpi -/- mice. ( A ) The neutrophil population was gated on the CD11bLY6G double-positive cells among the CD45-positive cells. ( B ) The macrophage population was gated on the CD64-positive cells among the CX3CR1-positive myeloid cells. Results from two independent experiments were pooled and shown here. ( C–E ) Expression levels of C-X-C motif chemokine receptor 2 ( Cxcr2 , C ), monocyte chemoattractant protein 1 ( Mcp-1 , D ), and C-C motif chemokine receptor 2 ( Ccr2 , E ) were assessed in the tibiotarsal tissue using RT-qPCR. ( F ) The serum cytokine profile was assessed using mouse cytokine/chemokine 32-plex array. An increase in IL-6 was observed in the infected Slpi -/- mice. ( G, H ) The serum level of neutrophil elastase (NE) was measured using an ELISA kit. ( I ) Serum levels of MMPs were assessed using a mouse MMP 5-Plex Discovery Assay. An increase in MMP-8 was observed in the infected Slpi -/- mice. Serum was obtained by cardiac puncture of WT and Slpi -/- C57BL/6 mice with/without infection between 21 and 24 dpi ( F , G , and I ) and of infected C3H/HeN mice at 21 dpi ( H ). black, PBS-sham infection; red, B. burgdorferi infection. Each data point represents an individual animal. The error bar represents mean ± SEM, and p-values were calculated using the nonparametric Mann–Whitney test. Figure 3—source data 1. Source data value for .

Article Snippet: The murine neutrophil elastase level was measured using the Mouse Neutrophil Elastase/ELA2 DuoSet ELISA (R&D Systems, #DY4517-05).

Techniques: Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Lung‐delivered IL‐10 mitigates Lung inflammation induced by repeated endotoxin exposures in male mice

doi: 10.14814/phy2.70253

Figure Lengend Snippet: Lung‐delivered IL‐10 administered after LPS exposures reduces repeated LPS‐induced weight loss and systemic inflammatory responses. (a) Mice were treated with LPS or saline daily for 3 days and received either treatment with IL‐10 (10 μg) or vehicle (veh) daily for 3 days administered 5 h after LPS or saline and were euthanized at 24 h after the final treatment. (b) Line graph depicts weights over time with scatter dot plot demonstrating percent change in weight upon study completion across indicated groups. (c) Acute phase protein pentraxin‐2, IL‐6, and neutrophil chemoattractant CXCL were quantified in serum across indicated groups. (d) Blood glucose levels and (e) peripheral blood neutrophils by percentage were quantified across indicated groups. All data expressed as mean with SD bars of n = 5 mice/saline exposed groups; n ≥ 8 mice/LPS exposed groups. Statistical significance by p ‐values versus Sal+Veh (no line) or denoted between groups by line.

Article Snippet: Levels of tumor necrosis factor (TNF)‐α (MTA00B‐1), transforming growth factor (TGF)‐β (DY1679), IL‐6 (M600B‐1), IL‐10 (M1000B‐1), the murine neutrophil chemoattractant CXCL1(MKC00B‐1), the murine leukocyte chemoattractants CCL2 (MJE00B) and CCL7 (ab205571), and the complement component C5a (DY2150) were quantitated by ELISA (R&D Systems) following manufacturer instructions.

Techniques: Saline