Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: Eyes of IL-6−/−, CXCL1−/−, and C57BL/6J mice were infected with 100 CFU B. cereus. Eyes were harvested and B. cereus were quantified. No significant differences in B. cereus burden were observed among mouse strains (P ≥ 0.163). Values represent means ± sem of n ≥ 6 eyes at each time point with at least 3 independent experiments.

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Infection

Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: Eyes of IL-6−/−, CXCL1−/−, and C57BL/6J mice were infected with 100 CFU B. cereus. Retinal function was assessed by ERG at 8 and 12 h postinfection. In infected eyes of IL-6−/− and CXCL1−/− mice, retained A- and B-wave responses were significantly greater than retained A- and B-wave responses in infected eyes of C57BL/6J mice. Values represent means ± sem of n ≥ 6 eyes each time point with at least 3 independent experiments. *P ≤ 0.0256

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Infection

Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: Eyes of IL-6−/−, CXCL1−/−, and C57BL/6J mice were infected with 100 CFU B. cereus. Uninfected and infected globes were harvested at 4, 8, and 12 h postinfection and processed for H&E staining. Uninfected C57BL/6J, IL-6−/− and CXCL1−/− had no inflammation and were architecturally and morphologically similar. At 4 h postinfection, there was fibrin in the anterior chamber and few inflammatory cells in the posterior segment in infected all eyes. At 8 and 12 h postinfection, infected eyes of C57BL/6J and IL-6−/− mice were similarly inflamed, with inflammatory cells in the posterior segment, dissolution of retinal layers, and retinal detachments. In contrast, infected eyes of CXCL1−/− mice had minimal inflammation and intact retinal layers at the same time points. Sections are representative of 3 eyes per time point with at least 3 independent experiments. Original magnification, ×10.

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Infection, Staining, Dissolution

Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: Eyes of IL-6−/−, CXCL1−/−, and C57BL/6J (C57) mice were infected with 100 CFU B. cereus. PMN infiltration in mouse eyes was estimated by quantifying MPO levels by using ELISA. MPO concentrations were similar in eyes from all groups at 4 h postinfection (P ≥ 0.38). MPO concentrations in infected eyes of IL-6−/− mice were similar at 8 h postinfection (P = 0.69) but were less than in eyes of C57BL/6J mice (P = 0.03) at 12 h postinfection. MPO concentrations in infected eyes of CXCL1−/− mice were less than that in infected eyes of C57BL/6J mice at 12 h postinfection (P ≤ 0.0001) and less than that in infected eyes of IL-6−/− mice at 8 and 12 h postinfection (P ≤ 0.009). Values represent means ± sem for n ≥ 5 eyes per group per time point with at least 3 independent experiments. *P ≤ 0.03.

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: Eyes of IL-6−/−, CXCL1−/−, and C57BL/6J mice were infected with 100 CFU B. cereus. Concentrations of TNF-α, IL-6, IL-1β, and CXCL1 were measured by using ELISA. Concentrations of IL-1β were similar in eyes of all mouse strains at all time points tested (P ≥ 0.17). Concentrations of TNF-α in infected eyes of C57BL/6J mice were similar to those in infected eyes of IL-6−/− mice at 8 and 12 h postinfection (P ≥ 0.14) but greater than that in infected eyes of CXCL1−/− mice at the same time points (P ≤ 0.004). CXCL1 concentrations in infected eyes of IL-6−/− mice were greater than that in infected eyes of C57BL/6J mice at 8 and 12 h postinfection, but the difference was only significant at 8 h postinfection (P = 0.03). IL-6 concentrations in infected eyes of CXCL1−/− mice were similar to that in eyes of C57BL/6J mice at all time points (P ≥ 0.57). Concentrations of IL-1β were similar in eyes of all mouse strains at all time points tested (P ≥ 0.17). Values represent means ± sem for n ≥ 5 eyes per group per time point with at least 3 independent experiments. *P ≤ 0.03.

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection

doi: 10.1189/jlb.3A0416-173R

Figure Lengend Snippet: C57BL/6J mice were injected with 100 CFU B. cereus alone (Untreated), B. cereus and 0.5 μg nonspecific isotype IgG2A (Isotype), or B. cereus and 125 ng/0.5 μl anti-CXCL1 antibody (Anti-CXCL1). All eyes were analyzed at 12 h postinfection. (A) B. cereus in the anti-CXCL1 treatment group were significantly less than in the isotype-treated and untreated groups (*P ≤ 0.011), but reduction was only 2-fold. (B) Retained A- and B-wave function was greater in anti-CXCL1 treated eyes than in untreated or isotype-treated eyes (*P ≤ 0.002). (C) Histologic sections showed that eyes of untreated and isotype-treated mice were similar. Eyes in these groups were highly inflamed and retinal architecture was lost. In contrast, there was less inflammation and intact retinal layers in infected eyes treated with anti-CXCL1 antibody. Original magnification, ×10. (D) MPO concentrations in infected eyes of mice treated with anti-CXCL1 antibody were significantly less than in untreated or isotype-treated eyes (*P ≤ 0.01). (E) Concentrations of all proinflammatory mediators in untreated and isotype-treated eyes were similar (P ≥ 0.05). Concentrations of TNF-α and CXCL1 were greater in untreated eyes than in anti-CXCL1 eyes (P ≤ 0.01), whereas concentrations of IL-6 and IL-1β were similar (P ≥ 0.11). Values represent means ± sem for n ≥ 5 eyes per group per time point with at least 3 independent experiments. *P ≤ 0.05.

Article Snippet: Anti-CXCL1 monoclonal antibody (125 ng/0.5 μl anti-KC/CXCL1 IgG 2A MAB 453, clone 48415; R&D Systems) was intravitreally injected with 100 CFU B. cereus [ 42 ].

Techniques: Injection, Infection

Journal: Nature Communications

Article Title: Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A

doi: 10.1038/s41467-022-34695-7

Figure Lengend Snippet: a – d Cremaster muscles of WT or IL-17A KO mice were stimulated with TNF (300 ng) or PBS for 4 h. Cremaster muscles were collected and immunostained for MCs (avidin), pericytes (α-SMA) and ICAM-1 or CXCL1. a – b Images of venules showing a ICAM-1 or b CXCL1 expression (pseudocolour intensity gradient) on pericytes (purple) and MCs (cyan), dashed lines the limit of MC, scale bars, 5 µm. c , d Quantification of c ICAM-1 MFI ( n = 6 WT, n = 5 IL-17A KO mice) and d CXCL1 MFI ( n = 5 mice) on pericytes. e , f Cremaster muscles of WT mice were stimulated with TNF (300 ng) or PBS in combination with a blocking anti-IL-17A or control mAbs (50 µg) for 4 h. e , f Quantification of e ICAM-1 MFI ( n = 6 PBS Ctrl mAb, n = 9 TNF Ctrl mAb, n = 6 PBS Anti-IL-17A mAb, n = 9 TNF anti-IL-17A mAb mice) and f CXCL1 MFI ( n = 4 PBS Ctrl mAb, n = 6 TNF Ctrl mAb, n = 4 PBS Anti-IL-17A mAb, n = 6 TNF anti-IL-17A mAb mice). Mean ± SEM (each mouse represents one independent experiment). c – f two-way ANOVA followed by Sidak’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to WT or ctrl mAb (ns = not significant). Source data are provided as a Source Data file.

Article Snippet: Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems.

Techniques: Muscles, Avidin-Biotin Assay, Expressing, Blocking Assay, Control

Journal: Nature Communications

Article Title: Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A

doi: 10.1038/s41467-022-34695-7

Figure Lengend Snippet: a – d TNF-stimulated or PBS-treated WT cremaster muscles were immunostained for MCs (avidin), pericytes (α-SMA) and ICAM-1 or CXCL1. a High magnification confocal image of a TNF-stimulated post-capillary venule (halved) showing enrichment of ICAM-1 (pseudocolour intensity) associated with pericytes (purple) in venule segment with or without MCs (cyan). White dashed line shows the limit of MC, scale bars, 5 µm. b , c Quantification of pericyte b ICAM-1 ( n = 11 PBS no MC, n = 10 PBS < 3 MCs, n = 9 > 3 MCs, n = 9 TNF no MC, n = 12 TNF < 3 MCs, n = 8 TNF > 3 MCs, * p value = 0.0451, ** p value = 0.0047, *** p value<0.0001, # p value = 0.0486) or c CXCL1 ( n = 12 PBS no MC, n = 13 PBS < 3 MCs, n = 7 > 3 MCs, n = 15 TNF no MC, n = 15 TNF < 3 MCs, n = 7 TNF > 3 MCs, * p value = 0.0411, ** p value = 0.0028, ** p value < 0.0024, # p value = 0.0087) MFI in venules without, less than or more than 3 MCs. d – f Quantification of pericyte ICAM-1 MFI in 5 µm-wide consecutive regions (as exemplified in a ) from a perivascular MC in d PBS and TNF treated WT mice ( n = 18 PBS, 31 TNF perivascular MC regions, data pooled from five mice, * p value = 0.0366, *** p value = 0.003, ## p value = 0.0044), e PBS and TNF treated IL-17A KO mice ( n = 36 perivascular MC regions, data pooled from five mice) or f in TNF treat e d mice in combination with ctrl mAb or anti-IL-17A mAb ( n = 27 Ctrl mAb, 28 anti-IL-17A mAb perivascular MC regions, * p value = 0.0265, # p value = 0.0133); data pooled from four mice. g – i Quantification of pericyte CXCL1 MFI in 5 µm-wide consecutive regions from a perivascular MC in g PBS and TNF-treated WT mice ( n = 31 PBS, 30 TNF perivascular MC regions, data pooled from five mice, * p value = 0.0322, *** p value = 0.003, ## p value = 0.0041), h PBS and TNF treated IL-17A KO mice ( n = 41 perivascular MC regions, data pooled from five mice) or i in TNF treated mice in combination with ctrl mAb or anti-IL-17A mAb ( n = 21 Ctrl mAb, 27 anti-IL-17A mAb perivascular MC regions * p value = 0.0248, # p value = 0.0448); data pooled from four mice. ICAM-1 and CXCL1 MFI were normalised to the most distal region from the MC (i.e., 15–20 µm). Mean ± SEM (each mouse represents one independent experiment). b – i two-way ANOVA followed by Sidak’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to PBS or 20–15 region or as indicated by # p < 0.05, ## p < 0.01 (ns = not significant). Source data are provided as a Source Data file.

Article Snippet: Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems.

Techniques: Muscles, Avidin-Biotin Assay

Journal: Nature Communications

Article Title: Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A

doi: 10.1038/s41467-022-34695-7

Figure Lengend Snippet: MC deficient animals were injected with BMMC from WT (MC WT ) or IL-17A KO (MC IL-17AKO ) mice. Four months later, MC-reconstituted mice were stimulated with TNF (300 ng) for 4 h. Cremaster muscles were collected and immunostained for neutrophils (MRP14), MCs (avidin), pericytes (α-SMA) and ICAM-1 or CXCL1. a Generation of MC-reconstituted mice. b Low-magnification image of the cremasteric microcirculation in MC WT and MC IL-17AKO mice depicting the reconstitution of MCs, scale bars 100 µm. c Number of perivascular MCs in MC WT and MC IL-17AKO mice ( n = 5 mice). d , e Quantification of d ICAM-1 and e CXCL1 MFI on pericytes ( n = 5 mice). f Images of post-capillary venules in TNF-treated MC WT and MC IL-17AKO mice showing increased expression of ICAM-1 in MC WT but not in MC IL-17AKO mice on pericytes, scale bars 5 µm. g Quantification of pericyte ICAM-1 MFI in regions at 5 µm interval from a perivascular MC in TNF-treated MC WT and MC IL-17AKO mice ( n = 28 MC WT , 27 MC IL-17AKO perivascular MC regions, data pooled from five mice, *** p value < 0.0001, ### p value < 0.0001). h Quantification of pericyte CXCL1 MFI in regions at 5 µm interval from a perivascular MC in TNF-treated MC WT and MC IL-17AKO mice, ( n = 19 MC WT , 27 MC IL-17AKO perivascular MC regions, data pooled from five mice, ** p value = 0.0088, ## p value = 0.0012). ICAM-1 and CXCL1 MFI were normalised to the more distal region (i.e., 15–20 µm). i Extravascular neutrophils in MC WT or MC IL-17AKO mice ( n = 5 mice, p value = 0.0094). j – k Correlation of the number of extravascular neutrophils and perivascular MCs in j MC WT ( n = 58 venules) and k MC IL-17AKO ( n = 60 venules); data pooled from five mice. Line indicated linear regression and dashed lines 95% confidence band. Mean±SEM (each mouse represents one independent experiment). c – e , i two-tailed Student’s t test; g , h two-way ANOVA followed by Sidak’s post-hoc test; j , k Spearman’s rank correlation test. **p < 0.01, ***p < 0.001 as compared to MC WT or 20-15 µm region or as indicated ## p < 0.01, ### p < 0.001 (ns = not significant). Source data are provided as a Source Data file.

Article Snippet: Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems.

Techniques: Injection, Muscles, Avidin-Biotin Assay, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A

doi: 10.1038/s41467-022-34695-7

Figure Lengend Snippet: Upon acute inflammation, perivascular MCs release the cytokine IL-17A that promotes localised enrichment of intercellular adhesion molecule 1 (ICAM-1) and chemokine CXCL1 on nearby pericytes from post-capillary venules. This response induces a graded expression of those the key molecules within the pericyte layer, allowing for the directed migration of neutrophils in the subendothelial space and subsequent exit from the vessel wall towards the nearest MCs.

Article Snippet: Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems.

Techniques: Expressing, Migration

Journal: The Journal of Neuroscience

Article Title: Heterozygous Gnal Mice Are a Novel Animal Model with Which to Study Dystonia Pathophysiology

doi: 10.1523/JNEUROSCI.1529-16.2017

Figure Lengend Snippet: List and dilution of primary and secondary antibodies used for immunoblotting and immunofluorescence

Article Snippet: IRDye 800CW-conjugated anti-mouse IgG , Rockland Immunochemical , 1:5000.

Techniques: Western Blot, Immunofluorescence