Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL7 contributes to angiotensin II‐ induced abdominal aortic aneurysm by promoting macrophage infiltration and pro‐inflammatory phenotype

doi: 10.1111/jcmm.16757

Figure Lengend Snippet: CCR1 antagonist‐BX471 suppressed CCL7‐induced macrophages migration and M1 polarization. (A and B) CCR1 protein level in (A) BMDMs incubated with rmCCL7(100 ng/ml) and (B) abdominal aortas of saline‐infused (n = 3) and Ang II‐infused (n = 3) mice. (C) Migration of BMDMs in response to rmCCL7 (100 ng/ml) and CCR1 antagonist‐BX471(10 μM) stimulation, BX471 was pre‐treated 1 hour before rmCCL7 incubation(n = 3). Scale bars represented 100 μm. (D) M1 markers (iNOS, IL‐6, IL‐12A, IL‐12B, TNF‐α) of BMDMs in response to rmCCL7 (100 ng/ml) and BX471(10 μM) stimulation, BX471 was pre‐treated 1 hour before rmCCL7 incubation(n = 3). Each plot represented 3 independent experiments. Values were represented as mean ± SEM; Student's t test was used in (A‐C). One‐way ANOVA was used for data analysis in (D). * P < .05; ** P < .01; *** P < .001, respectively

Article Snippet: Membranes were incubated with primary antibodies against iNOS (Cat# ab15323; Abcam), CD206 (Cat# ab64693; Abcam), CCR1 (Cat# MAB5986; R&D system), JAK2 (Cat# 3250s; CST), p‐JAK2 (Cat# 3771;CST), STAT1 (Cat# sc346; Santa Cruz), p‐STAT1 (Cat# 8826s; CST), GAPDH (Cat# KC‐5G5; KangChen bio‐tech) and tubulin (Cat# KC‐5T01; KangChen bio‐tech) overnight at 4oC and subsequently incubated with horseradish peroxidase (HRP) conjugated secondary antibodies which were detected by enhanced chemiluminescence (Cat# WBKLS0500; Millipore).

Techniques: Migration, Incubation, Saline

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL7 contributes to angiotensin II‐ induced abdominal aortic aneurysm by promoting macrophage infiltration and pro‐inflammatory phenotype

doi: 10.1111/jcmm.16757

Figure Lengend Snippet: CCL7‐CCR1 interaction regulated macrophage phenotype through JAK2 /STAT1 pathway. (A) Protein abundance of JAK2, p‐JAK2, STAT1, p‐STAT1 and p‐JAK2/JAK2, p‐STAT1/STAT1 ratio in BMDMs incubated with rmCCL7 (100 ng/ml) and pre‐treated 1 hour with CCR1 antagonist‐BX471(1 μM and 10 μM). (B) M1 markers (iNOS, IL‐6, IL‐12A, IL‐12B, TNF‐α) mRNA level in BMDMs incubated with rmCCL7 alone or in combination with JAK2 inhibitor‐Fedratinib (1 μM) and STAT1 inhibitor‐Fludarabine (1 μM). (C) Pathological mechanism diagram of CCL7 in Ang II‐induced AAA. Values were represented as mean ± SEM; One Way ANOVA was used for data analysis in (A‐B) * P < .05; ** P < .01; *** P < .001, respectively

Article Snippet: Membranes were incubated with primary antibodies against iNOS (Cat# ab15323; Abcam), CD206 (Cat# ab64693; Abcam), CCR1 (Cat# MAB5986; R&D system), JAK2 (Cat# 3250s; CST), p‐JAK2 (Cat# 3771;CST), STAT1 (Cat# sc346; Santa Cruz), p‐STAT1 (Cat# 8826s; CST), GAPDH (Cat# KC‐5G5; KangChen bio‐tech) and tubulin (Cat# KC‐5T01; KangChen bio‐tech) overnight at 4oC and subsequently incubated with horseradish peroxidase (HRP) conjugated secondary antibodies which were detected by enhanced chemiluminescence (Cat# WBKLS0500; Millipore).

Techniques: Quantitative Proteomics, Incubation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in normal, herpes simplex virus‐inoculated, but asymptomatic healthy (BDN) and Behçet's disease (BD) mice. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated from normal, BDN and BD mice. Frequencies of surface CCR1 + cells (a) and cytoplasm CCR1 + cells (b) of whole leukocytes were analyzed by FACS. Representative flow cytometric histograms of surface CCR1 + PBMC and lymph node cells are shown (c). (d) Protein expression of CCR1 by Western blot, (e) Expression ratio of CCR1 to GAPDH.

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Virus, Isolation, Expressing, Western Blot

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in cytokine‐treated normal and Behçet's disease (BD) mice. Interleukin‐10 (IL‐10) (25, 50 and 100 pg) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (50, 100 and 200 pg) were intraperitoneally injected into normal mice for five consecutive days and peripheral blood mononuclear cells (PBMC) were isolated. Frequencies of CCR1 + cells were analyzed by FACS for expression on the surface and in the cytoplasm of mice after treatment with IL‐10 (a) or GM‐CSF (b). IL‐10 (50 pg) and GM‐CSF (200 pg) were intraperitoneally injected into BD mice for five consecutive days. PBMC and lymph node cells were isolated. Frequencies of surface CCR1 + (c) and cytoplasm CCR1 + (d) PBMC and lymph node cells derived from IL‐10‐ and GM‐CSF‐treated BD mice were analyzed by FACS.

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Injection, Isolation, Expressing, Derivative Assay

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐treated normal mice. CCL3 (50 pg and 100 pg) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of PBMC and lymph node cells. Representative flow cytometric histograms of CCR1 + cells on the surface (a) and in the cytoplasm (b) of PBMC are shown in (c).

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐ and BX471‐treated Behçet's disease (BD) mice. CCL3 and BX471 were used to treat BD mice and frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of peripheral blood mononuclear cells (PBMC) and lymph node cells. Frequencies of CCR1 + cells in normal mice after treatment with BX471 are shown in (c) and (d).

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in colchicine (Col) and pentoxifylline (Pento) ‐treated normal and Behçet's disease (BD) mice. Colchicine (2 μg/mouse/day) and pentoxifylline (400 μg/mouse/day) were used to treat mice. Frequencies of surface and cytoplasm CCR1 + peripheral blood mononuclear cells (PBMC) and lymph node cells were analyzed in normal mice (a, b) and BD mice (c, d) by FACS. (e) Representative flow cytometric histogram of surface and cytoplasm CCR1 + PBMC following colchicine or pentoxifylline treatment of BD mice.

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated normal mice. Anti‐CCL3 antibody (1, 2, 20, 50 and 200 ng) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS.

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated Behçet's disease (BD) mice. Anti‐CCL3 antibody 20 ng was intraperitoneally injected into BD mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS. (c) The changes in disease severity score after CCL3 or anti‐CCL3 antibody treatment of BD mice. (d) Symptoms of BD mice after CCL3 or anti‐CCL3 antibody treatment.

Article Snippet: Antibody against CCR1 ( MAB5986 ) was purchased from R&D Systems and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (sc‐32233) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Injection, Isolation

Journal: bioRxiv

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

doi: 10.1101/2021.09.09.459638

Figure Lengend Snippet: Human (a) or mouse ( b ) neutrophils were examined for their chemotactic responses toward CCL3 (5ng/ml) after 1h exposure to glucose in normal (90 mg/dl) or diabetic range (200-500 mg/dl). ( c-g ) Neutrophils extracted from bone marrow of C57B were exposed to normal glucose (90 mg/dl) or high glucose (300 mg/dl) for 1h and assessed for CCR1 expression by Western blotting (c -d); for mRNA transcription analysis by RT-PCR ( e ); and for CCR1 surface expression (f-g) . ( h-i ) Neutrophils were purified from peripheral blood of normal (C57B) or diabetic (db/db) mice and assessed for the expression of CCR1 by Western blotting ( h ) and their respective densitometry values were plotted as the Mean ± SEM and shown in ( i ). ( j ) Equal number of neutrophils (extracted from Day 1 C57B and db/db wounds) were assessed by for the expression of CCR1 on neutrophils by flow cytometry (N=4; 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).

Article Snippet: Mouse CCR1 Alexa Fluor® 488-conjugated Antibody #FAB5986G-100UG (R & D Systems); Alexa Fluor® 700 anti-mouse NK-1.1 Antibody #108729 (BioLegend); Alexa Fluor® 700 anti-mouse CD3ε Antibody #152315 (BioLegend); Alexa Fluor® 700 anti-mouse CD19 Antibody #115527 (BioLegend); BV605 Hamster Anti-Mouse CD11c Clone HL3 (RUO) #563057 (BD Biosciences); LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation #L34966 (ThermoFisher Scientific); F4/80 antibody | Cl:A3-1 #MCA497PBT (Bio-Rad); BV650 Hamster Anti-Mouse CD11c Clone HL3 (RUO) #564079 (BD Biosciences); BV711 Rat Anti-Mouse CD45 Clone 30-F11 (RUO) #563709 (BD Biosciences); NK1.1 Monoclonal Antibody (PK136), PE, eBioscienceTM #12-5941-82 (ThermoFisher Scientific); CD19 Monoclonal Antibody (eBio1D3 (1D3)), PE, eBioscienceTM #12-0193-82 (ThermoFisher Scientific); CD3e Monoclonal Antibody (145-2C11), PE, eBioscienceTM #12-0031-82 (ThermoFisher Scientific); FPR1 Polyclonal Antibody #PA1-41398 (ThermoFisher Scientific); Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 #A-11037 (ThermoFisher Scientific); Ly6G Monoclonal Antibody (1A8-Ly6g), PE-Cyanine7, eBioscienceTM #25-9668-82 (ThermoFisher Scientific), PerCP Cy5.5 CD45 (BD Biosciences, Cat. 550994); APC Gr1, PE CD11b (BD Biosciences, Cat 553129); FITC CD69 (BD Biosciences, Cat #: 557392); and PECy7 F4/80 (Biolegends, Cat 123114).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Purification, Flow Cytometry

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in normal, herpes simplex virus‐inoculated, but asymptomatic healthy (BDN) and Behçet's disease (BD) mice. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated from normal, BDN and BD mice. Frequencies of surface CCR1 + cells (a) and cytoplasm CCR1 + cells (b) of whole leukocytes were analyzed by FACS. Representative flow cytometric histograms of surface CCR1 + PBMC and lymph node cells are shown (c). (d) Protein expression of CCR1 by Western blot, (e) Expression ratio of CCR1 to GAPDH.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Virus, Isolation, Expressing, Western Blot

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in cytokine‐treated normal and Behçet's disease (BD) mice. Interleukin‐10 (IL‐10) (25, 50 and 100 pg) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (50, 100 and 200 pg) were intraperitoneally injected into normal mice for five consecutive days and peripheral blood mononuclear cells (PBMC) were isolated. Frequencies of CCR1 + cells were analyzed by FACS for expression on the surface and in the cytoplasm of mice after treatment with IL‐10 (a) or GM‐CSF (b). IL‐10 (50 pg) and GM‐CSF (200 pg) were intraperitoneally injected into BD mice for five consecutive days. PBMC and lymph node cells were isolated. Frequencies of surface CCR1 + (c) and cytoplasm CCR1 + (d) PBMC and lymph node cells derived from IL‐10‐ and GM‐CSF‐treated BD mice were analyzed by FACS.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation, Expressing, Derivative Assay

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐treated normal mice. CCL3 (50 pg and 100 pg) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of PBMC and lymph node cells. Representative flow cytometric histograms of CCR1 + cells on the surface (a) and in the cytoplasm (b) of PBMC are shown in (c).

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐ and BX471‐treated Behçet's disease (BD) mice. CCL3 and BX471 were used to treat BD mice and frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of peripheral blood mononuclear cells (PBMC) and lymph node cells. Frequencies of CCR1 + cells in normal mice after treatment with BX471 are shown in (c) and (d).

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in colchicine (Col) and pentoxifylline (Pento) ‐treated normal and Behçet's disease (BD) mice. Colchicine (2 μg/mouse/day) and pentoxifylline (400 μg/mouse/day) were used to treat mice. Frequencies of surface and cytoplasm CCR1 + peripheral blood mononuclear cells (PBMC) and lymph node cells were analyzed in normal mice (a, b) and BD mice (c, d) by FACS. (e) Representative flow cytometric histogram of surface and cytoplasm CCR1 + PBMC following colchicine or pentoxifylline treatment of BD mice.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated normal mice. Anti‐CCL3 antibody (1, 2, 20, 50 and 200 ng) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated Behçet's disease (BD) mice. Anti‐CCL3 antibody 20 ng was intraperitoneally injected into BD mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS. (c) The changes in disease severity score after CCL3 or anti‐CCL3 antibody treatment of BD mice. (d) Symptoms of BD mice after CCL3 or anti‐CCL3 antibody treatment.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation

Journal: bioRxiv

Article Title: Maturation, developmental site, and pathology dictate murine neutrophil function

doi: 10.1101/2021.07.21.453108

Figure Lengend Snippet: a , PCA plot for Ly6G + , Ly6G Int and Ly6G Hi transcriptomic analysis from the peripheral blood, BM, spleen and liver of PBS-control and LPS-challenged mice. b, Quantification of Ly6G Int as %Ly6G + in the peripheral blood and spleen of naïve mice. Peripheral blood n=11 mice; spleen n=7 mice. Data were analysed by Mann-Whitney test. c , H&E identification of banded neutrophils in the splenic red pulp of naïve mice. Data representative of n=5 mice. Black arrows indicate banded neutrophils. d , Top 10 most enriched Process Networks from DEGs with increased expression in spleen Ly6G Int compared to spleen Ly6G Hi neutrophils from naive mice. Data from transcriptomic analysis. e , Heatmap showing row scaled expression for genes associated with neutrophil maturation (identified by Xie et al., 2020) for Ly6G Int and Ly6G Hi neutrophils in spleen. Data from transcriptomic analysis. f , Quantification of EdU + as %Ly6G Int and Ly6G Hi neutrophils in the BM of naïve mice. Ly6G Int and Ly6G Hi neutrophils n=5 mice at each time-point. Data were analysed by 2way ANOVA with Sidak’s multiple comparisons test. Blue star (*) indicates comparisons with p-value<0.0001. For ‘ns’ at 36hr time-point, p-value=0.9989. g , Quantification of EdU + as %Ly6G Int and Ly6G Hi neutrophils in the spleen of naïve mice. Ly6G Int and Ly6G Hi neutrophils n=5 mice at each time-point. Data were analysed by 2way ANOVA with Sidak’s multiple comparisons test. Blue star (*) indicates comparisons with p- value<0.0001. h , Quantification of EdU + as %CD101 - and CD101 + neutrophils in the spleen of naïve mice. CD101 - and CD101 + neutrophils n=5 mice at each time-point. Data were analysed by 2way ANOVA with Sidak’s multiple comparisons test. Blue star (*) indicates comparisons with p-value<0.0001. i , Heatmap showing row scaled expression for genes associated with neutrophil maturation (identified by Xie et al., 2020) for Ly6G Int neutrophils in BM and spleen from naïve mice. Data from transcriptomic analysis. j , Quantification of CCR1 + , CCR3 + , CXCR1 + and CXCR2 + as %Ly6G Int neutrophils in the BM and spleen of naïve mice. Data from flow cytometry analysis. BM Ly6G Int n=5 mice; spleen Ly6G Int n=5 mice. Data were analysed by 2way ANOVA with Sidak’s multiple comparisons test. k , Top 10 most enriched Process Networks from DEGs with increased expression in spleen Ly6G Int compared to BM Ly6G Int neutrophils from naive mice. Data from transcriptomic analysis. l , Correlation for genes identified in process networks from between spleen Ly6G Int and BM Ly6G Hi , shown as log2 fold change (FC) compared to BM Ly6G Int . Data from transcriptomic analysis. m , Heatmap showing column scaled expression for genes with high expression unique to spleen Ly6G Int neutrophils. Data from transcriptomic analysis. Bulk Ly6G + , Ly6G Int and Ly6G Hi neutrophil RNA-Seq data in , , , . PBS: peripheral blood Ly6G Hi n=4 mice; BM Ly6G Int n=4 mice; BM Ly6G Hi n=4 mice; spleen Ly6G Int n=6 mice; spleen Ly6G Hi n=7 mice; liver Ly6G + n=7 mice. LPS: peripheral blood Ly6G Int n=4 mice; peripheral blood Ly6G Hi n=4 mice; BM Ly6G Int n=3 mice; BM Ly6G Hi n=3 mice; spleen Ly6G Int n=4 mice; spleen Ly6G Hi n=4 mice; liver Ly6G + n=4 mice. Dots in , , represent individual mice. Error bars in , , represent mean±SEM.

Article Snippet: The following antibodies were used: CD45 (clone 30-F11), CD11b (clone M1/70), Ly6G (clone 1A8), CD3e (clone 145-2C11), CD19 (clone 6D5), Nkp46 (clone 29A1.4), Ter119 (clone TER119) ICAM-1 (clone YN1/1.7.4), CD115 (clone AFS98), CD117 (clone 2B8), Sca-1 (clone D7), CCR6 (clone 29-2L17), CD48 (clone HM48-1), Ki-67 (clone 16A8), CD16/32 (clone 93), CD18 (clone M18/2), CXCR4 (clone L276F12), CXCR2 (clone SA045E1), Streptavidin (all Biolegend), Ly6B.2 (clone 7/4, Bio-Rad), CD133 (clone 13A4, eBioscience), CD49d (clone R1-2, ThermoFisher), CD101 (clone Moushi101, ThermoFisher), Siglec-F (clone E50-2440, BD Biosciences), CCR1 (clone 643854, Bio-Techne).

Techniques: MANN-WHITNEY, Expressing, Flow Cytometry, RNA Sequencing Assay