Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: TLR2 −/− , NOD2 −/− , FPR1 −/− and wt mice were inoculated intradermally with S. aureus . (A) Mean total lesion size (cm 2 ) ± SEM. (B) In vivo bioluminescence quantified by mean total flux (photons/s) ± SEM (logarithmic scale). (C) Mean IL-1β protein levels (pg/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. (D) Mean myeloperoxidase (MPO) activity (U/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. * p<0.05, † p<0.01, TLR2 −/− , NOD2 −/− or FPR1 −/− mice versus wt mice (Student's t -test). Data are from 2 experiments with at least 4 mice/group per experiment.

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: In Vivo, Activity Assay

Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: Neutrophils from mouse bone marrow were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. IL-1β protein levels (mean ± SEM) were measured in culture supernatants by ELISA. (A, B) IL-1β protein levels in supernatants from S. aureus -infected neutrophils from TLR2 −/− , NOD2 −/− , FPR1 −/− , ASC −/− and wt mice. (C, D) IL-1β protein levels in supernatants from wt mouse neutrophils infected with S. aureus (C) or MRSA (D), in the absence and presence of an NLRP3-inhibitor (glibenclamide), a caspase-1 inhibitor (Z-YVAD-FMK) or anti-staphylococcal α-toxin antiserum. The respective vehicle controls (DMSO or normal rabbit IgG) for the inhibitors had IL-1β protein levels that did not differ than media alone (data not shown). Data are from 3–5 mice per group. *p<0.05; † p<0.01, ‡ p<0.001, TLR2 −/− , NOD2 −/− , FPR1 −/− or ASC −/− mice versus wt mice (A, B) or the NLRP3-inhibitor, caspase-1 inhibitor or anti-staphylococcal α-toxin antiserum treatment versus media alone (C, D) (Student's t -test).

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Advanced Healthcare Materials

Article Title: A Comparative Inflammation‐on‐a‐Chip with a Complete 3D Interface: Pharmacological Applications in COPD‐Induced Neutrophil Migration

doi: 10.1002/adhm.202301673

Figure Lengend Snippet: A comparative inflammation‐on‐a‐chip model to measure neutrophil transendothelial migration. a) Neutrophil transendothelial migration can be measured in plasma samples from healthy subjects and COPD patients using a single device, therefore enabling comparative analysis. b) A schematic illustration of neutrophil and its representative surface receptors. Neutrophils express the CD11b (integrin alpha M), complement component 5a receptor (C5aR), leukotriene B4 receptor (LTB4R), CXCR1 (IL8RA), CXCR2 (IL8RB), and formyl peptide receptors 1 and 2 (FPR1 and FPR2). The Figure was created using BioRender.com.

Article Snippet: The cells were then stained with fluorescently conjugated primary antibodies, anti‐CXCR1‐APC (Cat. No. 320 612), anti‐CXCR2‐PE‐Cy7 (Cat. No. 320 716), anti‐CD11b‐BV605 (Cat. No. 301 332), anti‐C5aR‐PerCP‐Cy5 (Cat. No. 344 312), anti‐ICAM‐1 (Cat. No. 353 108), anti‐CD62E (Cat. No. 336 012), anti‐CD62P (Cat. No. 304 920) (BioLegend, San Diego, CA, USA), anti‐LTB4R‐FITC (Cat. No. FAB099F), anti‐FPR1‐APC (Cat. No. FAB3744A), or anti‐FPR2‐PE (Cat. No. FAB3744A) (R&D Systems) in FACS buffer containing 2% (v/v) BSA and 2 mM EDTA in DPBS for 30 min at 4 °C.

Techniques: Migration

Journal: Scientific Reports

Article Title: The hypothesis that Helicobacter pylori predisposes to Alzheimer’s disease is biologically plausible

doi: 10.1038/s41598-017-07532-x

Figure Lengend Snippet: MKN-28 mRNAs levels of FPR1, FPRL1, FPRL2 and CTSG. Results are representative of three independent experiments. Each value is the mean ± SD of three replicas. Expression values were normalized against the human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene. Stability assay, carried out using the BestKeeper tool, indicated that GAPDH was more stable then ACT-β at 30 (1.03 vs 2.89) and 60 min (1.89 vs 3.29). Statistical analysis was carried out with the GraphPad Prism version 5.03 (GraphPad, La Jolla, CA, USA). Differences in expression levels between 30 min and 1 h are all significantly different (P < 0.001); a Hp(2-20) concentration was 2 × 10 −5 M; b Hpgb = H. pylori growth broth (140 µl/well).

Article Snippet: Expression levels of the FPR1 (Hs04235426_s1), FPRL1 (Hs02759175_s1), FPRL2 (Hs00266666_s1), and CTSG (Hs01113415_g1) were measured by rt-PCR using the TaqMan PCR master 2X reagent (Applied Biosystem) and the Applied Biosystem iCycler according to the manufacturer’s protocol.

Techniques: Expressing, Stability Assay, Concentration Assay

Journal: Scientific Reports

Article Title: The hypothesis that Helicobacter pylori predisposes to Alzheimer’s disease is biologically plausible

doi: 10.1038/s41598-017-07532-x

Figure Lengend Snippet: Pathways represented among the 77 genes associated with AD and differentially expressed upon activation with Hp(2-20).

Article Snippet: Expression levels of the FPR1 (Hs04235426_s1), FPRL1 (Hs02759175_s1), FPRL2 (Hs00266666_s1), and CTSG (Hs01113415_g1) were measured by rt-PCR using the TaqMan PCR master 2X reagent (Applied Biosystem) and the Applied Biosystem iCycler according to the manufacturer’s protocol.

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: The hypothesis that Helicobacter pylori predisposes to Alzheimer’s disease is biologically plausible

doi: 10.1038/s41598-017-07532-x

Figure Lengend Snippet: Profiles of 65 AD genes catalogued in the AlzBase database.

Article Snippet: Expression levels of the FPR1 (Hs04235426_s1), FPRL1 (Hs02759175_s1), FPRL2 (Hs00266666_s1), and CTSG (Hs01113415_g1) were measured by rt-PCR using the TaqMan PCR master 2X reagent (Applied Biosystem) and the Applied Biosystem iCycler according to the manufacturer’s protocol.

Techniques:

Journal: eLife

Article Title: Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound

doi: 10.7554/eLife.72071

Figure Lengend Snippet: ( a–b ) Neutrophils were isolated from the peripheral blood of C57BL/6 and db/db animals to assess: ( a ) their ability to chemotax toward 100 nM fMLP, or ( b ) for the expression of FPR1 by Western blotting. ( c ) Densitometry values associated with ( b ) are plotted as Mean ± SEM (N = 4 blood pools/group, each blood pool was from 4 mice). ( d ) Equal number of neutrophils (isolated from Day 1 C57B and db/db wounds) were assessed for the surface expression of FPR1 on neutrophils by flow cytometry (N = 3 mice/group). ( e–f ) Purified neutrophils from peripheral blood of non-diabetic individuals ( e ), or C57BL/6 bone marrow ( f ), were exposed to media containing glucose in normal range (90 mg/dl) or in diabetic range (200–500 mg/dl) for 1 hr to assess their ability to chemotax toward 100 nM fMLP. Data are plotted as Mean ± SEM. (N > 4). ( g–h ) Neutrophils from C57BL/6 bone marrow were exposed to glucose in normal range (90 mg/dl) or in diabetic range (300 mg/dl) for 1 hr and assessed for surface expression of FPR1 by flow cytometry. A representative histogram is shown in ( g ) and the corresponding tabulated data, plotted as Mean ± SEM is shown in ( h ) (N = 3). ( i–j ) Murine neutrophils (from C57B bone marrow) were exposed to glucose in normal or diabetic range (90 mg/dl or 300 mg/dl) for 1 hr and assessed for the expression of indicated proteins by Western blotting. Representative Western blots are shown in ( i ) and corresponding densitometry values, plotted as Mean ± SEM, are shown in ( j ). (N ≥ 3 independent experiments). ( k–m ) Murine neutrophils exposed to normal or diabetic glucose, as described for ( g–h ), were assessed for Cyclic AMP production by ELISA ( k ), and for mRNA of Fpr1 and Plcγ by RT-PCR ( l-m ). (N ≥ 3, ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses between groups were conducted by One-way ANOVA with additional post hoc testing, and pair-wise comparisons between groups were performed or by unpaired Student’s t -test). Figure 2—source data 1. Related to . Figure 2—source data 2. Related to . Figure 2—source data 3. Related to . Figure 2—source data 4. Related to . Figure 2—source data 5. Related to . Figure 2—source data 6. Related to . Figure 2—source data 7. Related to . Figure 2—source data 8. Related to . Figure 2—source data 9. Related to . Figure 2—source data 10. Related to . Figure 2—source data 11. Related to . Figure 2—source data 12. Related to . mRNA data for Plcγ by RT-PCR.

Article Snippet: Antibody , Anti-FPR1 antibody(Rabbit polyclonal) , NOVUS Biological , Cat# NB100-56473, RRID: AB_838228 , WB (1:1000).

Techniques: Isolation, Expressing, Western Blot, Flow Cytometry, Purification, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound

doi: 10.7554/eLife.72071

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-FPR1 antibody(Rabbit polyclonal) , NOVUS Biological , Cat# NB100-56473, RRID: AB_838228 , WB (1:1000).

Techniques: Staining, Sequencing, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Recombinant, Software, SYBR Green Assay

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells.

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: Fig. 5 | SNPs in human FPR1 associated with neutrophil resistance to the Y. pestis T3SS. a, Quantification of Y. pestis KIM D27(pMM83) translocation of YopM–Bla in neutrophils from five donors (1–5) compared to U937 cells. b, Quantification of migrating neutrophils from donors 2 and 4 following addition of fMLF (1–100 nM) or priming with mock, Y. pestis KIM D27(WT) or KLD29(ΔlcrV) (2 × 107 CFU ml−1). c, Model illustrating the position of amino acid substitutions in human FPR1. d, Surface display of FPR1 revealed by immunofluorescence microscopy using Alexa-488-labelled anti-FPR1 antibodies (left). DAPI staining showing nuclei of U937 cells and variants (right). One of three repeats is shown. e, Quantification of migrating U937, FPR1−/−, FPR1−/−(FPR1) and FPR1−/−(FPR1(R190W)) cells in a Transwell assay primed with mock, fMLF, Y. pestis KIM D27(WT) or KLD29(ΔlcrV). One of three repeats is shown (a, b, d, e). Data are mean + s.e.m. (n = 3 biological replicates, shown as circles) (a, b, e). One-way ANOVA and Bonferroni post hoc analyses (a, e) or two-tailed t-test (b) were used to identify significant differences. ***P < 0.001; NS, not significant.

Article Snippet: Plasmid encoding FPR1 (FPR1 sgRNA-resistant allele), which expresses human FPR1 from the SRα promoter18, was obtained from Addgene (plasmid 62600).

Techniques: Translocation Assay, Immunofluorescence, Microscopy, Staining, Transwell Assay, Two Tailed Test