Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 1. After M1 MΦs phagocytose NETs, P-SEP produced within M1 MΦs is released into extracellular space over time. (a) Cell morphology image of M1 MΦ phagocytosing PMA-NETs. (MG stain, magnification 1,000 ×). Scale bar: 10 µm. (b) Immunofluorescence staining results of M1 MΦ phagocytosing PMA-NETs. NETs indicated by arrows are citrullinated histone H3-positive. Scale bar: 10 µm. (c) Enlarged view of M1 MΦ phagocytosing PMA-NETs. Arrows indicate images of P-SEP production by M1 MΦs phagocytosing NETs. Scale bar: 5 µm. (d, e) After co-culturing M1 MΦs and NETs, cells were collected over time and analyzed for intracellular P-SEP expression using flow cytometry. P-SEP levels in culture medium supernatant were measured. (f) Western blotting of intracellular P-SEP expression intensity and P-SEP at 0, 15, and 180 min after NET phagocytosis by M1 MΦs. P-SEP protein levels were quantified using Image J. β-actin band intensity was used to correct P-SEP band intensity. The unprocessed western blot image is shown in Supplementary Fig. S11. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± standard deviation (SD; n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Produced, Staining, Immunofluorescence, Expressing, Flow Cytometry, Western Blot, Two Tailed Test, Standard Deviation

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 2. M1 MΦs phagocytose NETs and produce P-SEP. (a) P-SEP production after co-culture of M1 MΦs with PMA-NETs and DH5α-NETs was compared. (b) P-SEP production after co-culture of M1 MΦs or monocytes with PMA-NETs was compared. (c) Correlation between P-SEP production and NET ratio after M1 MΦ phagocytosed NETs. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Co-Culture Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 3. Evaluation of P-SEP levels in inhibiting NET formation, M1 MΦ phagocytosis, and protease inhibition. (a) Evaluation of culture medium supernatant P-SEP levels after adding various inhibitors. (b) Western blotting of P-SEP protein levels in M1 MΦs after adding various inhibitors. P-SEP protein levels were quantified using Image J. β-actin band intensity was used to correct P-SEP band intensity. The unprocessed western blot image is shown in Supplementary Fig. S12. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Inhibition, Western Blot, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 5. Identification of organs with MΦs producing P-SEP in a mouse model of sepsis. (a) Ly6c-positive cells were compared with positivity rate of Ly6C-positive M1 MΦs in each organ as M1 MΦs. M1 MΦs were the most abundant in the lungs of mice in the sham group, with a small number of M1 MΦs in the liver, spleen, and kidneys. Compared to the sham group, mice in the CLP group showed significantly increased Ly6C positivity in each organ, especially markedly in the lungs and liver. Each dot represents one mouse. (b) Comparing P-SEP expression in M1 MΦs in each organ between sham and CLP groups, intracellular P-SEP expression in each organ was lower in sham group mice, while P-SEP expression in M1 MΦs in lungs, liver, and spleen was significantly increased in CLP group mice. Each dot represents one mouse. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 5). **p < 0.01. P-SEP, presepsin; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Expressing, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 4. Evaluated blood NET ratio and blood P-SEP production in CLP-treated sepsis model mice. (a) Gross cecum findings for the sham group and CLP-treated sepsis model mice. As indicated by arrows, the ceca of CLP-treated mice were necrotic. (b, c) Comparison of viable bacteria calculated from colony counts. Each dot represents one mouse. (d, e) Blood NET ratio and P-SEP levels were evaluated in CLP-treated sepsis model mice. Each dot represents one mouse. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 5). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Comparison, Bacteria, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 6. Images of P-SEP production in MΦ of lungs, liver, and kidneys. Immunofluorescence staining for P-SEP in MΦs in each organ showed that only a few MΦs in the sham group were positive for P-SEP, but P-SEP was markedly positive in MΦs from the lungs, liver, and kidney in the CLP group. Scale bar: 10 µm. P-SEP, presepsin; M1 MΦs, M1 macrophage; CLP, cecal ligation and puncture.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Immunofluorescence, Staining, Ligation

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 7. Schematic diagram of the mechanism by which tissue MΦ produces P-SEP to phagocytose NETs. When bacteria infect blood vessels, neutrophils release NETs for biological defense. When MΦs in the lungs, liver, and spleen phagocytose increase NETs in the body, P-SEP is produced, and blood P-SEP levels are high. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Bacteria, Produced

Journal: mSphere

Article Title: Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi , and its role in host cell adhesion

doi: 10.1128/msphere.00403-23

Figure Lengend Snippet: Knockout of HcaA reduced H. cinaedi cytotoxicity and adherence to cells. (A) LDH assay to evaluate cytotoxicity. Each cell was infected with wild-type (MRY08-1234 WT or MRY12-0027 WT) and HcaA-knockout (MRY08-1234 ΔHcaA or MRY12-0027 ΔHcaA) strains for 24 h (multiplicity of infection [MOI], 100). Cytotoxicity (%) was calculated using the method described in the instructions. (B–D) Adhesion and invasion assays against Caco-2 cells. Caco-2 cells were infected with wild-type (MRY08-1234 WT or MRY12-0027 WT) and HcaA knockout (MRY08-1234 ΔHcaA or MRY12-0027 ΔHcaA) strains for 4 h (MOI 100). The adhesion rate (B) and invasion rate (C) were calculated by dividing the number of adherent bacterial cells and invading bacterial cells, respectively, by the total number of infected bacterial cells. The invasion rate of adherent bacteria (D) was calculated by dividing the number of invading bacteria cells by the number of adherent bacteria cells. Results are presented as the mean ± standard deviation (SD) of six independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns: not specified.

Article Snippet: Immediately after the addition of bacteria, the plates were centrifuged at 300 × g for 5 min at 25°C, followed by incubation in 5% CO 2 humidified atmosphere, at 37°C for 4 h. Cytotoxicity was evaluated by measuring lactate dehydrogenase using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Tokyo, Japan).

Techniques: Knock-Out, Lactate Dehydrogenase Assay, Infection, Bacteria, Standard Deviation