Journal: EBioMedicine

Article Title: Circulating growth/differentiation factor 15 is associated with human CD56 bright natural killer cell dysfunction and nosocomial infection in severe systemic inflammation

doi: 10.1016/j.ebiom.2019.04.018

Figure Lengend Snippet: Reduced expression of IL-12Rβ2 and phosphorylation of STAT4 on/in CD56 bright NK cells in systemic inflammation. PBMC from patients and from healthy control subjects (group 1; n = 10) were stimulated with inactivated S. aureus in the presence of autologous serum (a, b, d–f) or FCS (c). (a) Representative dot plots for staining of IL-12Rβ2 on CD56 bright NK cells from a control subject and a patient 1 d after injury. Numbers indicate the percentage of positive cells. (b) Cumulative data of the percentage of IL-12Rβ2 + CD56 bright NK cells in the presence of autologous serum. Note, due to insufficient cell numbers only n = 8–9 values were available for d1 and d8. (c) Percentage of IL-12Rβ2 + CD56 bright NK cells in the presence of FCS (n = 8 on d8). (d) Representative histograms showing the phosphorylated STAT4 (pSTAT4) expression of unstimulated (none) and stimulated cells from one control subject and one patient on day 1 after injury. ΔMFI depicts the difference between the mean fluorescence intensity (MFI) of stimulated and unstimulated cells. (e) Cumulative data of the ΔMFI of CD56 bright NK cells from controls and patients 1 or 8 d after injury (each n = 8) or at the day of discharge presented as Tukey box plots. Horizontal lines indicate the median and interquartile range. (f) Spearman correlation between the percentage of IL-12Rβ2 + cells (obtained from b) and ΔMFI pSTAT4 (obtained from e). Statistical analyses were performed using the Mann Whitney U test. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus controls. c, control subjects; D, day of discharge; iso, isotype control.

Article Snippet: Intracellular staining for phosphorylated STAT4 and Smad1/8 proteins was performed after 18 h of culture using antibodies against phosphorylated STAT4 (pY693; clone 38/p-Stat4, PE-labelled, BD Biosciences), phosphorylated Smad1/8 (cross-reactive with pSmad5; clone N6-1233, PE-labelled, BD Biosciences), and the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) as described previously [ ].

Techniques: Expressing, Staining, Fluorescence, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Circulating growth/differentiation factor 15 is associated with human CD56 bright natural killer cell dysfunction and nosocomial infection in severe systemic inflammation

doi: 10.1016/j.ebiom.2019.04.018

Figure Lengend Snippet: The suppressive effect of the patients' sera on CD56 bright NK cell-derived IFN-γ synthesis correlates with a diminished IL-12Rβ2 expression. PBMC from healthy donors were stimulated with inactivated S. aureus in the presence of 10% pooled sera from healthy control subjects or with serum from individual patients obtained on day 1 and day 8 after injury (group 1; n = 10). (a) Representative dot plots of IL-12Rβ2 staining. Numbers indicate the percentage of IL-12Rβ2 + CD56 bright NK cells. (b) Cumulative data of the IL-12Rβ2 expression in the presence of individual sera. The value of the percentage of IL-12Rβ2 + CD56 bright NK cells obtained in the presence of the pooled sera from healthy control subjects was set as 100% (indicated as dotted line). (c) Spearman correlation between the expression of IL-12Rβ2 (as shown in b) and the production of IFN-γ (as shown in b). (d) Cumulative data of the activation of STAT4 (pSTAT4) in the presence of individual sera. The value of the ΔMFI in CD56 bright NK cells obtained in the presence of the pooled sera from control subjects was set as 100% (indicated as dotted line). Horizontal lines indicate the median and interquartile range of individual values. Statistical analyses were performed using the Wilcoxon signed rank test. (e) Isolated monocytes from healthy donors were cultured in the pooled serum from control subjects or from patients 8 d after injury (group 1). The cells were stimulated with inactivated S. aureus alone or together with recombinant IFN-γ and the release of IL-12 was quantified. Bars show mean + SD of triplicate cultures and are representative for 3 separate experiments. Statistically significant differences between control sera and the patients' sera were tested using unpaired Student's t -test. ***, p < 0.001*, p < 0.05; ##, p < 0.01 versus serum pool of control subjects.

Article Snippet: Intracellular staining for phosphorylated STAT4 and Smad1/8 proteins was performed after 18 h of culture using antibodies against phosphorylated STAT4 (pY693; clone 38/p-Stat4, PE-labelled, BD Biosciences), phosphorylated Smad1/8 (cross-reactive with pSmad5; clone N6-1233, PE-labelled, BD Biosciences), and the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) as described previously [ ].

Techniques: Derivative Assay, Expressing, Staining, Activation Assay, Isolation, Cell Culture, Recombinant

Journal: Frontiers in Immunology

Article Title: Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

doi: 10.3389/fimmu.2021.785072

Figure Lengend Snippet: CGRP and SAX, but not CGRP 1–8 , increase STAT4 expression in MDLCs. (A, B) PHA/IL2-activated PBMCs were serum-starved overnight at 37°C, and left untreated (Un) or stimulated for 30 min with either IL12 or IFNα. Shown are representative Western blots (of n = 4 independent experiments using PBMCs from different individuals) of total STAT4 (A) and pSTAT4 (B) expression. (C, D) MDLCs were cytokine-starved overnight at 37°C, and treated with CGRP (0.1 μM), SAX (0.1 μM), CGRP 1–8 (10 μM) or LPS (10 μg/ml) as positive control. In panel (C) , shown is a representative Western blot (of n = 4 independent experiments using MDLCs from different individuals) of total STAT4 expression. In panel (D) , shown are mean ± SEM folds expression of total STAT4, normalized to that of beta actin. *p < 0.0500, **p < 0.0050, two-sided Student’s t-test. (E) MDLCs were cytokine-starved overnight at 37°C and treated with CGRP (0.1 μM) or SAX (0.1 μM). The CGRP receptor antagonist BIBN4096 (BIBN, 1 μM) was added 15 min before addition of agonists. Shown are mean ± SEM (of n = 4 independent experiments using MDLCs from different individuals) folds expression of total STAT4, normalized to that of beta actin. (F) MDLCs were treated as described in panels (C, D) above, and further stimulated for 30 min with combination of IL12 + IFNα. Shown is a representative Western blot (of n = 4 independent experiments using MDLCs from different individuals) of pSTAT4 expression.

Article Snippet: The blots were next incubated overnight at 4°C with commercial rabbit polyclonal Abs suitable for WB, directed against human STAT4 (Proteintech #13028-1AP, 0.5 μg/ml) or phosphorylated STAT4 (pSTAT4; R&D systems, #AF4319, 1 μg/ml), followed by 1:1,000 dilution of HRP-conjugated donkey-anti-rabbit IgG Ab (Southern Biotech) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Positive Control

Journal: Frontiers in Immunology

Article Title: Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

doi: 10.3389/fimmu.2021.785072

Figure Lengend Snippet: Summary of the requirements of CGRP receptor activation for inhibition of mucosal HIV-1 transmission. (1) In LCs, HIV-1 binding to langerin induces viral internalization and subsequent degradation, while virions escaping degradation trans-infect CD4+ T-cells. (2) We previously showed that CGRP activates its cognate receptor expressed by LCs and affects a multitude of cellular and molecular process (not shown), resulting in significant inhibition of mucosal HIV-1 trans-infection in-vitro and ex-vivo . (3) We show in the present study that SAX, a long-acting metabolically stable analogue of CGRP, also activates the CGRP receptor. (4) Both CGRP and SAX increase expression of langerin (not shown) and STAT4 (that can be readily phosphorylated upon subsequent cytokine stimulation), which result in inhibition of HIV-1 trans-infection in-vitro and ex-vivo . (5) In contrast, several CGRP peptide fragments fail to activate the CGRP receptor and to increase langerin/STAT4 expression, and accordingly lack anti-HIV-1 activity. (6) CGRP-mediated inhibition of HIV-1 dissemination from LCs to CD4+ T-cells might permit their long-term maintenance in the BLT model of mucosal HIV-1 infection in-vivo .

Article Snippet: The blots were next incubated overnight at 4°C with commercial rabbit polyclonal Abs suitable for WB, directed against human STAT4 (Proteintech #13028-1AP, 0.5 μg/ml) or phosphorylated STAT4 (pSTAT4; R&D systems, #AF4319, 1 μg/ml), followed by 1:1,000 dilution of HRP-conjugated donkey-anti-rabbit IgG Ab (Southern Biotech) for 1 h at room temperature.

Techniques: Activation Assay, Inhibition, Transmission Assay, Binding Assay, Infection, In Vitro, Ex Vivo, Metabolic Labelling, Expressing, Activity Assay, In Vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Up-regulation of the IL-12 receptor beta 2 chain in Crohn's disease.

doi: 10.4049/jimmunol.165.12.7234

Figure Lengend Snippet: FIGURE 3. Expression of both active (phosphorylated) and inactive STAT4 proteins in mucosal samples from two CD (lanes 1–4) and two UC (lanes 5–8) patients and two non-IBD controls (lanes 9 and 10). In both patients with CD and UC, mucosal samples were taken from involved and uninvolved tissue. Total proteins were immunoprecipitated (IP) with STAT4 Ab, run on 10% SDS-PAGE under reducing conditions, and im- munoblotted with anti-phosphotyrosine Ab (p-Tyr). After detection of p- Tyr-STAT4 proteins, the membrane was stripped and reblotted with an- other STAT4 Ab to ascertain equivalent loading of the lanes. A marked phosphorylation of STAT4 is seen in affected mucosal samples from CD compared with UC or non-IBD controls. The example is representative of three separate experiments.

Article Snippet: After p-Tyr analysis, blots were stripped by incubation for 30 min at 50°C in stripping medium (2% SDS, 0.05 M Tris, and 0.1 M 2-ME) and then incubated with Ab against STAT4 (H-119; Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, SDS Page, Membrane, Phospho-proteomics