Journal: International Journal of Molecular Sciences

Article Title: CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

doi: 10.3390/ijms232416149

Figure Lengend Snippet: CCL4 expression in airway epithelial cells. ( A ) Immunofluorescence analysis of nasal polyps (NP) with rich eosinophils (Eos) (n = 5) or poor Eos (n = 5), or without Eos (n = 5). CCL4 and EpCAM expression levels were measured. CCL4 (green), EpCAM (orange), MBP (red), and the nucleus (blue) are stained with hematoxylin and eosin (HE) (i). Images were obtained using an FV3000 confocal microscope (100 × objectives). The scale bars in the bottom-right corner indicate 100 μm. CCL4 intensity is expressed as a fold change relative to EpCAM (ii). The correlation of CCL4 expression with eosinophil count in NPs (iii) and Eos from the peripheral blood (iv). ( B ) CCL4 , CCL5 , CCL11 , and CCL26 mRNA levels were determined in NPs and uncinate process tissues (UTs) obtained from patients with ECRS. ( C , D ) CCL4 expression in BEAS-2B cells co-cultured with purified Eos (from peripheral blood) overnight. CCL4 , CCL5 , CCL11 , and CCL26 mRNA levels were determined ( C ). Images (CCL4, green; nucleus, blue) were obtained using an FV3000 confocal microscopes (200 × objectives) ( D ). Scale bars in the bottom-right corner indicate 10 μm. The values in B and C represent the mean ± SEM of four experiments. # p < 0.05, ## p < 0.01 (vs. NPs with rich Eos in A, UTs from the same patients in B, and NPs without Eos in C).

Article Snippet: The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described.

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Cell Culture, Purification

Journal: International Journal of Molecular Sciences

Article Title: CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

doi: 10.3390/ijms232416149

Figure Lengend Snippet: Effect of CCL4 on eosinophil accumulation in OVA-induce allergic model. Before each OVA challenge, recombinant murine CCL4 was intranasally administrated. ( A ) Total and differential cell counts in BALF. ( B , C ) Eosinophil infiltration in nasal tissues. HE staining ( B ) and the number of eosinophils infiltrated into the nasal tissues ( C ) are shown. Eosinophil infiltrates are indicated using black arrowheads. The scale bars in the bottom-right corner indicate 10 μm. The values represent the mean ± SEM of four ( A ) or three ( C ) experiments. * p < 0.05 (between the two groups). The experimental design is detailed in the Materials and Methods section (4.9. Sensitization and airway challenge).

Article Snippet: The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described.

Techniques: Recombinant, Staining

Journal: International Journal of Molecular Sciences

Article Title: CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

doi: 10.3390/ijms232416149

Figure Lengend Snippet: CCL4-mediated eosinophil activation. ( A ) Purified peripheral blood eosinophils coincubated with BEAS-2B cells were treated overnight with human recombinant CCL4. CD69 expression in eosinophils was evaluated [(i) percentage of CD69+ Siglec-8+ double-positive cells and (ii) CD69 median fluorescence intensity (MFI) ratio to isotype control]. ( B ) CCL4 mRNA (i) and protein (ii) expression in eosinophils coincubated with BEAS-2B cells. ( C ) CCR5 mRNA (i) and protein (ii) expression in eosinophils coincubated with BEAS-2B cells. ( D ) Eosinophils pretreated with maraviroc (MVC)—a CCR5 antagonist—were stimulated with CCL4. The values represent the mean ± SEM of four experiments. * p < 0.05, ** p < 0.01 (between the two groups).

Article Snippet: The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described.

Techniques: Activation Assay, Purification, Recombinant, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

doi: 10.3390/ijms232416149

Figure Lengend Snippet: CCL4-mediated eosinophil activation signaling. ( A ) Phosphorylation levels of multiple kinases in eosinophils. Purified peripheral blood eosinophils were treated with CCL4 (10 μg/mL) for 5 min using a Human Phospho-Kinase Antibody Array Kit (R&D). Phosphorylation levels of platelet-derived growth factor receptor (PDGFR)β, Src kinase family (Lck, Src, and Yes), extracellular signal-regulated kinase (ERK), and control are presented as dots (left panel) or a relative value to vehicle (right panel). ( B ) Colocalization of CCR5, PDGFRβ, and Src in eosinophils. CCR5 (red), PDGFRβ (green or pink), Src (light blue), and the nucleus (blue) are shown. Images were obtained using an FV3000 confocal microscope (600 × objectives). The scale bars in the bottom-right corner indicate 10 μm. The results are representative of at least three experiments. ( C ) Purified peripheral blood eosinophils were pretreated with imatinib—a PDGFRβ inhibitor (I, 5 μM) —and/or dasatinib—a Src inhibitor (D, 10 nM) —for 30 min, followed by overnight incubation with CCL4 (10 μg/mL). CD69 expression in eosinophils was determined [(i) percentage of CD69+ Siglec-8+ double-positive cells and (ii) CD69 median fluorescence intensity (MFI) ratio to isotype control]. The values represent the mean ± SEM of four experiments. # p < 0.05 (vs. nontreatment control); * p < 0.05 (between the two groups).

Article Snippet: The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described.

Techniques: Activation Assay, Purification, Ab Array, Derivative Assay, Microscopy, Incubation, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

doi: 10.3390/ijms232416149

Figure Lengend Snippet: Mechanism of CCL4-mediated transactivation in the eosinophil signaling pathway. CCR5—a transmembrane protein coupled to G proteins that is stimulated with CCL4—induces PDGFRβ transactivation via tyrosine phosphorylation of Src, which leads to biological activities in eosinophils via signal transduction, such as kinase (e.g., ERK) phosphorylation.

Article Snippet: The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described.

Techniques: Transduction

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Mixed neuronal-glial cerebrocortical cultures from WT or IFNAR1KO mice were incubated with IFNβ in increasing doses (from 500 to 5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h. Total RNA was extracted from cell lysates, analyzed by qRT-PCR and normalized to GAPDH expression levels. ( a–d ) RNA expression is shown as fold change (FC) in relation to vehicle treated controls which were defined as baseline activity. ( e–h ) Time course for protein expression measured in cell-free supernatants for CCL3, CCL4, CCL5 and CXCL10 using a commercially available multiplex assay as described in Methods. Baseline protein expression in vehicle treated cell cultures is represented as 0 h time point. Values are mean ± s.e.m.; n = 3–5 independent experiments per ISG; ***p < 0.001, **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test. For clarity, the significance is only indicated for differences between treatments and baseline within each experimental group.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Quantitative RT-PCR, Expressing, RNA Expression, Activity Assay, Multiplex Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Mixed neuronal-glial cerebrocortical cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL (200 pM) and mouse IFNβ (5,000 U/ml) in the presence and absence of neutralizing antibodies against CCL3, CCL4, CCL5, IFNγ or CXCL10. IFNγ antibody was used as control for neutralizing antibodies since this protein was undetectable in cerebrocortical cell cultures. ( b ) Mouse cerebrocortical cultures from IFNAR1KO mice were stimulated with gp120 BaL for 24 h the presence or absence of mouse IFNβ (5,000 U/ml) or BSA/PBS control. ( c ) Cerebrocortical cell cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL in the presence and absence of murine CCL4 (2 or 20 nM). Neuronal survival was assessed by immunofluorescence microscopy and counting of MAP-2/NeuN double-positive neurons. Values are mean ± s.e.m.; n = 3–5 independent experiments with 3–7 replicates and an average of 9,000 (IFNAR1KO) or 5,700 (WT) cells counted per condition; ** p < 0.01, *** p < 0.001 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Control, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: RNA was purified from one brain hemisphere each of 4–5 month-old HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle and analyzed by qRT-PCR for fold-change (FC) in ISG expression. Significant changes in gene expression were observed between IFNβ and vehicle treatment groups in WT brains ( a ) for CCL4, and in gp120tg brains ( b ) for CCL4, CXCL11 and IRF3. Expression of transgenic HIVgp120 was not affected by IFNβ ( c ). Values are mean ± s.e.m.; n = 4–5 animals per group/genotype; *p < 0.05, student’s t-test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Purification, Quantitative RT-PCR, Expressing, Gene Expression, Transgenic Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Sagittal brains sections of HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle (veh) were immunolabeled for CCL4, neuronal MAP-2 or astrocytic GFAP. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was labeled with Hoechst (H) 33342. The fluorescence-labeled brain sections were analyzed using confocal laser-scanning microscopy. Representative images of cortex layer III are shown; scale bar, 50 μm.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Immunolabeling, Labeling, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Cerebrocortical cultures from mice were prepared to either contain microglia, neurons and astrocytes (M + N + A) or were depleted of microglia (N + A) or neurons and microglia (A). Complete and depleted cell cultures were incubated with mIFNβ (5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h and concentrations of CCL4 were measured in cell-free supernatants using a commercially available multiplex assay as described in Methods. Maximum concentrations were reached in samples of 12 to 24 h mIFNβ exposure and compared to vehicle-treated, baseline samples. Values are mean ± s.e.m.; n = 3 independent experiments; *p < 0.05, student’s t-test. ( b ) Microglia-depleted rat cerebrocortical cultures were exposed for 24 h to 50% cell-free conditioned media (CM) from human MDM in the presence or absence of human IFNβ (5,000 U/ml). MDM were previously stimulated for 24 h with HIV-1 gp120 BaL (MDM gp120 CM) or vehicle (MDM CM). Following the incubation the cells were fixed and permeabilized. Neurons were immunolabeled for neuronal MAP-2 and NeuN and nuclear DNA was stained with H33342. Neuronal survival was assessed using fluorescence microscopy and cell counting as described in Methods. Values are mean ± s.e.m.; n = 2 independent experiments, with 4–8 replicates and an average of 4,000 cells counted per condition; **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Multiplex Assay, Immunolabeling, Staining, Fluorescence, Microscopy, Cell Counting

Journal: Journal of Inflammation Research

Article Title: Identifying Therapeutic Targets and Potential Drugs for Diabetic Retinopathy: Focus on Oxidative Stress and Immune Infiltration

doi: 10.2147/JIR.S500214

Figure Lengend Snippet: Candidate Drug Predicted Using DSigDB

Article Snippet: Primary antibodies targeting CCL4 (1:1000, Bioss, bs-2475R, China), FCGR2B (1:1000, Abclonal, A12553, China) and β-actin (1:1000, ZSGB-BIO, TA-09, China) overnight at 4°C.

Techniques:

Journal: Journal of Inflammation Research

Article Title: Identifying Therapeutic Targets and Potential Drugs for Diabetic Retinopathy: Focus on Oxidative Stress and Immune Infiltration

doi: 10.2147/JIR.S500214

Figure Lengend Snippet: Docking Results of Available Proteins With Candidate Drugs

Article Snippet: Primary antibodies targeting CCL4 (1:1000, Bioss, bs-2475R, China), FCGR2B (1:1000, Abclonal, A12553, China) and β-actin (1:1000, ZSGB-BIO, TA-09, China) overnight at 4°C.

Techniques: Binding Assay

Journal: Journal of Inflammation Research

Article Title: Identifying Therapeutic Targets and Potential Drugs for Diabetic Retinopathy: Focus on Oxidative Stress and Immune Infiltration

doi: 10.2147/JIR.S500214

Figure Lengend Snippet: Molecular docking simulation diagram. ( A ) (+)-chelidonine binds to CCL4. ( B ) oxazolone binds to CCL4. ( C ) eugenol binds to CCL4. ( D ) simvastatin binds to CCL4. ( E ) (+)-chelidonine binds to FCGR2B. ( F ) oxazolone binds to FCGR2B. ( G ) eugenol binds to FCGR2B. ( H ) AGN-PC-0JHFVD binds to FCGR2B. ( I ) AGN-PC-0JHFVD binds to FOXP3. ( J ) simvastatin binds to FOXP3. CCL4 is marked in red, FCGR2B in wheat, FOXP3 in green, and candidate drugs in cyan. Blue solid lines represent hydrogen bonds, grey dotted lines represent hydrophobic interactions, and green dotted lines represent π-stacking interactions.

Article Snippet: Primary antibodies targeting CCL4 (1:1000, Bioss, bs-2475R, China), FCGR2B (1:1000, Abclonal, A12553, China) and β-actin (1:1000, ZSGB-BIO, TA-09, China) overnight at 4°C.

Techniques:

Journal: Journal of Inflammation Research

Article Title: Identifying Therapeutic Targets and Potential Drugs for Diabetic Retinopathy: Focus on Oxidative Stress and Immune Infiltration

doi: 10.2147/JIR.S500214

Figure Lengend Snippet: External validation of CCL4 and FCGR2B. ( A ) The mRNA levels of CCL4 and FCGR2B were evaluated in animal models by RT-qPCR. ( B ) The protein levels of CCL4 and FCGR2B were evaluated animal models by WB. ** represents P <0.01, * represents P <0.05 and ns, no significance.

Article Snippet: Primary antibodies targeting CCL4 (1:1000, Bioss, bs-2475R, China), FCGR2B (1:1000, Abclonal, A12553, China) and β-actin (1:1000, ZSGB-BIO, TA-09, China) overnight at 4°C.

Techniques: Quantitative RT-PCR