Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) Peripheral blood samples from 6 SARS-CoV-2–infected survivors were collected longitudinally during hospitalization, as described previously . Concentrations of plasma CXCL12 and peripheral blood LDNs were measured. Black dotted line: Average plasma CXCL12 of healthy donors. Red dotted line: Average LDNs of healthy donors. ( B ) Correlation between concentration of plasma CXCL12 and percentage of peripheral blood LDNs analyzed by repeated measures correlation (with log transformation to Ln to meet linear assumption). R = –0.5437174 ( P = 0.003374754). ( C ) Peripheral blood samples from 9 SARS-CoV-2–infected deceased patients were collected at multiple time points during hospitalization, as described previously . Concentrations of plasma CXCL12 and peripheral blood LDNs are shown. Black dotted line: Average CXCL12 of healthy donors. Red dotted line: Average LDNs of healthy donors. ( D ) Correlation between concentration of plasma CXCL12 and percentage of peripheral blood LDNs analyzed by repeated measures correlation (with log transformation to Ln to meet linear assumption). R = –0.01767992 ( P = 0.9184839).

Article Snippet: The binding capability of α-CXCL12 antibodies (clone MAB310 and MCX120) was determined by ELISA using reagents and standard samples included in the ELISA Kit (MCX120, R&D Systems).

Techniques: Infection, Clinical Proteomics, Concentration Assay, Transformation Assay

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) The correlation between the concentration of plasma CXCL12 and the fold change in peripheral blood CD4 + and CD8 + T cells, immature neutrophils, degranulated neutrophils, and LDNs in SARS2-N501Y MA30 –infected mice (5000 PFU) on day 5 after infection ( n = 8). R = 0.003954 ( P = 0.8824) (CD4 + T cells), 0.0006628 ( P = 0.9518) (CD8 + T cells), 0.1851 ( P = 0.2873) (immature neutrophils), 0.02186 ( P = 0.7268) (degranulated neutrophils), and 0.9547 ( P < 0.0001) (LDNs). Data are representative of 3 independent experiments. ( B ) Expression of CXCR4 by peripheral blood CD4 + and CD8 + T cells, and neutrophil subsets of mice infected with SARS2-N501Y MA30 on day 5 after infection. ( C ) Expression of intracellular CXCL12 in CD45 – CD31 + CD54 + vascular endothelial cells on day 5 after infection. ( D ) Summary of CXCL12 expression (mean fluorescence intensity, MFI) in peripheral blood cell subsets and endothelial cells, n = 5. Data are representative of 2 independent experiments and are mean ± SEM. ** P < 0.01 by 1-way ANOVA with Tukey’s multiple comparisons. ( E ) RNA (right y axis) and protein (left y axis) CXCL12 levels in homogenates of bone marrow harvested from SARS2-N501Y MA30 –infected mice were determined at the indicated time points by RT-qPCR and ELISA, respectively. n = 4. Data are representative of 2 independent experiments.

Article Snippet: The binding capability of α-CXCL12 antibodies (clone MAB310 and MCX120) was determined by ELISA using reagents and standard samples included in the ELISA Kit (MCX120, R&D Systems).

Techniques: Concentration Assay, Clinical Proteomics, Infection, Expressing, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A – D ) Experimental setup ( A ), survival ( B ), lung histopathology ( C ), and infectious viral titers ( D ) of 8- to 10-month-old C57BL/6N mice infected with 1000 PFU SARS2-N501Y MA30 followed by treatment with α-CXCL12 antibody or its isotype control (IC, isotype Ig). Data in B are a summary of 4 independent experiments ( n = 20). Data in C are representative images and a summary of 2 independent experiments ( n = 10, samples harvested on day 5 after infection). Data in D are mean ± SEM ( n = 8) and are a summary of 2 independent experiments. LOD, limit of detection. Scale bar: 430 μm. ( E – G ) Experimental setup ( E ), numbers of total neutrophils/LDNs ( F ), and CFSE-stained neutrophils/LDNs ( G ) identified in peripheral blood, lung, and bone marrow (BM) after treatment with α-CXCL12 antibody or IC ( n = 5). Data are mean ± SEM and are representative of 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by t-test in F and G . ( H and I ) Experimental setup ( H ) and survival ( I ) of 8- to 10-month-old C57BL/6N mice infected with 1000 PFU SARS2-N501Y MA30 followed by treatment with α-CXCL12 antibody and α-Ly6G antibody or IC. Data in I are a summary of 2 independent experiments ( n = 8).

Article Snippet: The binding capability of α-CXCL12 antibodies (clone MAB310 and MCX120) was determined by ELISA using reagents and standard samples included in the ELISA Kit (MCX120, R&D Systems).

Techniques: Histopathology, Infection, Control, Staining

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) Weights of mice infected with 500 PFU IAV-PR8 ( n = 6), 500 PFU MERS MA ( n = 6), or 5000 PFU SARS2-N501Y MA30 ( n = 8) measured on day 5 after infection. ( B and C ) Correlation between plasma CXCL12 concentration and fold change in peripheral blood neutrophils ( B ) and LDNs ( C ) in IAV-PR8–, MERS MA -, and SARS2-N501Y MA30 –infected mice on day 5 after infection. ( B ) R = 0.04474 ( P = 0.6874) (IAV-PR8), 0.02521 ( P = 0.7639) (MERS MA ), and 0.5072 ( P = 0.0475) (SARS2-N501Y MA30 ). ( C ) R = 3.492 × 10 –6 ( P = 0.9972) (IAV-PR8), 0.4028 ( P = 0.1759) (MERS MA ), and 0.9547 ( P < 0.0001) (SARS2-N501Y MA30 ). Data are representative of 2 independent experiments. Mock- ( D ), SARS2-N501Y MA30 –infected (5000 PFU) ( E ), or IAV-PR8–infected (500 PFU) ( F ) middle-aged C57BL/6N mice (8–10 months old, n = 5/group), or MERS MA -infected (500 PFU) hDPP4 -KI mice ( G ) were treated with 0.5 mg/kg body weight of MERS (EMC)-NTD-Fc, SARS-2 (N501Y)-RBD-Fc, or SARS-2 (ancestral)-RBD-Fc in 0.5 mL PBS by i.v. injection on days 2 and 4 after infection. Mice were euthanized on day 5 after infection and abdominal aortas were harvested. The expression of CXCL12 in endothelial cells was determined by intracellular staining via flow cytometry. Data are representative of 2 independent experiments. ( H and I ) SARS2-N501Y MA30 –infected (5000 PFU) mice were treated with SARS2 (N501Y)-RBD-Fc ( n = 8) or control MERS (EMC)-NTD-Fc ( n = 5). Survival ( H ) and endothelial cell expression of CXCL12 ( I ) were determined. **** P < 0.01 by 1-way ANOVA with Tukey’s multiple comparisons. Data are mean ± SEM and are representative of 2 independent experiments. mpk, mg/kg body weight.

Article Snippet: The binding capability of α-CXCL12 antibodies (clone MAB310 and MCX120) was determined by ELISA using reagents and standard samples included in the ELISA Kit (MCX120, R&D Systems).

Techniques: Infection, Clinical Proteomics, Concentration Assay, Injection, Expressing, Staining, Flow Cytometry, Control

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) Peripheral blood samples from 6 SARS-CoV-2–infected survivors were collected longitudinally during hospitalization, as described previously . Concentrations of plasma CXCL12 and peripheral blood LDNs were measured. Black dotted line: Average plasma CXCL12 of healthy donors. Red dotted line: Average LDNs of healthy donors. ( B ) Correlation between concentration of plasma CXCL12 and percentage of peripheral blood LDNs analyzed by repeated measures correlation (with log transformation to Ln to meet linear assumption). R = –0.5437174 ( P = 0.003374754). ( C ) Peripheral blood samples from 9 SARS-CoV-2–infected deceased patients were collected at multiple time points during hospitalization, as described previously . Concentrations of plasma CXCL12 and peripheral blood LDNs are shown. Black dotted line: Average CXCL12 of healthy donors. Red dotted line: Average LDNs of healthy donors. ( D ) Correlation between concentration of plasma CXCL12 and percentage of peripheral blood LDNs analyzed by repeated measures correlation (with log transformation to Ln to meet linear assumption). R = –0.01767992 ( P = 0.9184839).

Article Snippet: For intracellular cytokine staining, lymphocytes were cultured in 96-well dishes at 37°C for 5–6 hours in the presence of 2 μM peptide pools and brefeldin A (BD Biosciences), labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with PE α-mouse CXCL12 (clone MAB310, R&D Systems), APC α-mouse IFN-γ (clone XMG1.2, BioLegend), and FITC α-mouse TNF (clone MP6-XT22, BioLegend) antibodies (1:100 dilution).

Techniques: Infection, Clinical Proteomics, Concentration Assay, Transformation Assay

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) The correlation between the concentration of plasma CXCL12 and the fold change in peripheral blood CD4 + and CD8 + T cells, immature neutrophils, degranulated neutrophils, and LDNs in SARS2-N501Y MA30 –infected mice (5000 PFU) on day 5 after infection ( n = 8). R = 0.003954 ( P = 0.8824) (CD4 + T cells), 0.0006628 ( P = 0.9518) (CD8 + T cells), 0.1851 ( P = 0.2873) (immature neutrophils), 0.02186 ( P = 0.7268) (degranulated neutrophils), and 0.9547 ( P < 0.0001) (LDNs). Data are representative of 3 independent experiments. ( B ) Expression of CXCR4 by peripheral blood CD4 + and CD8 + T cells, and neutrophil subsets of mice infected with SARS2-N501Y MA30 on day 5 after infection. ( C ) Expression of intracellular CXCL12 in CD45 – CD31 + CD54 + vascular endothelial cells on day 5 after infection. ( D ) Summary of CXCL12 expression (mean fluorescence intensity, MFI) in peripheral blood cell subsets and endothelial cells, n = 5. Data are representative of 2 independent experiments and are mean ± SEM. ** P < 0.01 by 1-way ANOVA with Tukey’s multiple comparisons. ( E ) RNA (right y axis) and protein (left y axis) CXCL12 levels in homogenates of bone marrow harvested from SARS2-N501Y MA30 –infected mice were determined at the indicated time points by RT-qPCR and ELISA, respectively. n = 4. Data are representative of 2 independent experiments.

Article Snippet: For intracellular cytokine staining, lymphocytes were cultured in 96-well dishes at 37°C for 5–6 hours in the presence of 2 μM peptide pools and brefeldin A (BD Biosciences), labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with PE α-mouse CXCL12 (clone MAB310, R&D Systems), APC α-mouse IFN-γ (clone XMG1.2, BioLegend), and FITC α-mouse TNF (clone MP6-XT22, BioLegend) antibodies (1:100 dilution).

Techniques: Concentration Assay, Clinical Proteomics, Infection, Expressing, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A – D ) Experimental setup ( A ), survival ( B ), lung histopathology ( C ), and infectious viral titers ( D ) of 8- to 10-month-old C57BL/6N mice infected with 1000 PFU SARS2-N501Y MA30 followed by treatment with α-CXCL12 antibody or its isotype control (IC, isotype Ig). Data in B are a summary of 4 independent experiments ( n = 20). Data in C are representative images and a summary of 2 independent experiments ( n = 10, samples harvested on day 5 after infection). Data in D are mean ± SEM ( n = 8) and are a summary of 2 independent experiments. LOD, limit of detection. Scale bar: 430 μm. ( E – G ) Experimental setup ( E ), numbers of total neutrophils/LDNs ( F ), and CFSE-stained neutrophils/LDNs ( G ) identified in peripheral blood, lung, and bone marrow (BM) after treatment with α-CXCL12 antibody or IC ( n = 5). Data are mean ± SEM and are representative of 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by t-test in F and G . ( H and I ) Experimental setup ( H ) and survival ( I ) of 8- to 10-month-old C57BL/6N mice infected with 1000 PFU SARS2-N501Y MA30 followed by treatment with α-CXCL12 antibody and α-Ly6G antibody or IC. Data in I are a summary of 2 independent experiments ( n = 8).

Article Snippet: For intracellular cytokine staining, lymphocytes were cultured in 96-well dishes at 37°C for 5–6 hours in the presence of 2 μM peptide pools and brefeldin A (BD Biosciences), labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with PE α-mouse CXCL12 (clone MAB310, R&D Systems), APC α-mouse IFN-γ (clone XMG1.2, BioLegend), and FITC α-mouse TNF (clone MP6-XT22, BioLegend) antibodies (1:100 dilution).

Techniques: Histopathology, Infection, Control, Staining

Journal: The Journal of Clinical Investigation

Article Title: CXCL12 ameliorates neutrophilia and disease severity in SARS-CoV-2 infection

doi: 10.1172/JCI188222

Figure Lengend Snippet: ( A ) Weights of mice infected with 500 PFU IAV-PR8 ( n = 6), 500 PFU MERS MA ( n = 6), or 5000 PFU SARS2-N501Y MA30 ( n = 8) measured on day 5 after infection. ( B and C ) Correlation between plasma CXCL12 concentration and fold change in peripheral blood neutrophils ( B ) and LDNs ( C ) in IAV-PR8–, MERS MA -, and SARS2-N501Y MA30 –infected mice on day 5 after infection. ( B ) R = 0.04474 ( P = 0.6874) (IAV-PR8), 0.02521 ( P = 0.7639) (MERS MA ), and 0.5072 ( P = 0.0475) (SARS2-N501Y MA30 ). ( C ) R = 3.492 × 10 –6 ( P = 0.9972) (IAV-PR8), 0.4028 ( P = 0.1759) (MERS MA ), and 0.9547 ( P < 0.0001) (SARS2-N501Y MA30 ). Data are representative of 2 independent experiments. Mock- ( D ), SARS2-N501Y MA30 –infected (5000 PFU) ( E ), or IAV-PR8–infected (500 PFU) ( F ) middle-aged C57BL/6N mice (8–10 months old, n = 5/group), or MERS MA -infected (500 PFU) hDPP4 -KI mice ( G ) were treated with 0.5 mg/kg body weight of MERS (EMC)-NTD-Fc, SARS-2 (N501Y)-RBD-Fc, or SARS-2 (ancestral)-RBD-Fc in 0.5 mL PBS by i.v. injection on days 2 and 4 after infection. Mice were euthanized on day 5 after infection and abdominal aortas were harvested. The expression of CXCL12 in endothelial cells was determined by intracellular staining via flow cytometry. Data are representative of 2 independent experiments. ( H and I ) SARS2-N501Y MA30 –infected (5000 PFU) mice were treated with SARS2 (N501Y)-RBD-Fc ( n = 8) or control MERS (EMC)-NTD-Fc ( n = 5). Survival ( H ) and endothelial cell expression of CXCL12 ( I ) were determined. **** P < 0.01 by 1-way ANOVA with Tukey’s multiple comparisons. Data are mean ± SEM and are representative of 2 independent experiments. mpk, mg/kg body weight.

Article Snippet: For intracellular cytokine staining, lymphocytes were cultured in 96-well dishes at 37°C for 5–6 hours in the presence of 2 μM peptide pools and brefeldin A (BD Biosciences), labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with PE α-mouse CXCL12 (clone MAB310, R&D Systems), APC α-mouse IFN-γ (clone XMG1.2, BioLegend), and FITC α-mouse TNF (clone MP6-XT22, BioLegend) antibodies (1:100 dilution).

Techniques: Infection, Clinical Proteomics, Concentration Assay, Injection, Expressing, Staining, Flow Cytometry, Control