Journal: Frontiers in Immunology

Article Title: Skin repair and immunoregulatory effects of myeloid suppressor cells from human cord blood in atopic dermatitis

doi: 10.3389/fimmu.2023.1263646

Figure Lengend Snippet: Immunobiological characterization of human umbilical cord blood (hUCB) myeloid-derived suppressor cells (MDSCs) in vitro using human T cells. (A) The proliferation- and differentiation-stage CD33 + CD11b + MDSC markers during the 6-week culture period of hUCB-MDSCs. Flow cytometric analysis of hUCB-MDSCs stained with individual MDSC-positive or -negative surface marker antibodies. (CD33 + CD11b + ≥ 70%, CD14 + ≥ 90%) (B) The expression of immune suppressive molecules by hUCB-MDSCs was analyzed using FITC anti-inducible nitric oxide synthase (iNOS)2 antibody, PE anti-indoleamine 2,3-deoxygenase (IDO) antibody, and PerCP-Cy5.5 anti-arginase-1 (ARG1) antibody staining. Cells were analyzed using a FACS Lyric device after two washes in stain buffer and incubation on ice for 30 min. Black, isotype control antibody. Red, each specific antibody. (iNOS ≥ 200 MFI, IDO ≥ 450 MFI, ARG1 ≥ 400 MFI) (C) hUCB-MDSC-mediated immune suppression of T cells, stimulated by anti-CD3/CD28 beads for 6 days in human peripheral blood mononuclear cells (PBMCs), was tested at a PBMC–hUCB-MDSC ratio of 1:1. (Suppression ≥ 45%). (D) hUCB-MDSC-mediated immunoregulation of human CD4 + T-cell differentiation, stimulated using Th2 or Th17 differentiation kits for 13 or 6 days in human CD4 + T cells, was tested at a human CD4 + T cells–hUCB-MDSC ratio of 1:1. * p < 0.05, ** p < 0.01.

Article Snippet: ARG1 (Nor-NOHA 0.5 mM; Selleckchem, Houston, TX, USA) or iNOS inhibitor (1400W 0.1 mM; Medchem Express, Monmouth, NJ, USA) and hUCB-MDSCs was simultaneously treated.

Techniques: Derivative Assay, In Vitro, Staining, Marker, Expressing, Incubation, Control, Cell Differentiation

Journal:

Article Title: IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis

doi: 10.1172/JCI25141

Figure Lengend Snippet: IL-6 is necessary and sufficient to induce injury of oligodendrocytes and axons in spinal cord organotypic cultures by generating NO. (A) CSF (100 μl) from a TM or control patient was added to culture media of spinal cord organotypic cultures, and cellular injury was assessed by ethidium homodimer uptake with Hoechst counterstain (inset). IL-6 was immunodepleted by preincubating TM CSF with an IL-6 antibody and clearing the IL-6 antibody complex with protein A sepharose. Magnification, ×20. (B) Tissue lysates from spinal cord organotypics were generated at various times after the administration of IL-6 and subjected to SDS-PAGE followed by immunoblot analysis. (C) Quantification of the data shown in B by chemiluminescent signal intensity of 3 independent experiments. (D) RT-PCR analysis of RNA derived from spinal cord organotypic cultures at the indicated times after addition of IL-6 at 2,000 pg/ml. (E) Dual-color confocal microscopy was carried out with spinal cord organotypic cultures treated with IL-6 for 24 hours. Microglia were identified by incubating live cultures with DiI-Ac-LDL, which is endocytosed by phagocytosing cells. After fixation, iNOS immunohistochemistry was carried out, revealing the expression of iNOS within microglia. Scale bar: 50 μm. (F) NT and iNOS preferentially accumulated within the exterior white matter of spinal cord organotypic cultures. Scale bar: 200 μm. (G) Addition of IL-6 to a final concentration of 2,000 pg/ml to spinal cord organotypic cultures in the presence or absence of the iNOS inhibitor 1400W. *P < 0.05. (H) WT and iNOS-heterozygous and -KO spinal cord organotypic cultures were exposed to IL-6 and assessed for cellular death.

Article Snippet: Recombinant rat IL-6 (catalog no. 557008; BD Biosciences — Pharmingen) was made as a 2 μg/ml stock in 1 mg/ml BSA in PBS and used at final concentrations of 500 pg/ml and 2,000 pg/ml. iNOS inhibitor 1400W dihydrochloride (catalog no. ALX-270-073; Alexis Biochemicals) was made at 100 mM stock in water and used at a final concentration of 100 μM.

Techniques: Generated, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Confocal Microscopy, Immunohistochemistry, Expressing, Concentration Assay

Journal:

Article Title: IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis

doi: 10.1172/JCI25141

Figure Lengend Snippet: PARP activation is necessary for IL-6–induced toxicity. (A) PARP activity of IL-6–treated spinal cord (SC) organotypic cultures was assessed in the presence or absence of the PARP inhibitor 4-ANI or the iNOS inhibitor 1400W. (B) Spinal cord organotypic cultures were incubated with IL-6 in the presence or absence of the PARP inhibitor 4-ANI, and the amount of NT accumulation was assessed as a marker of iNOS activity. (C) Spinal cord organotypic cultures were incubated with IL-6 in the presence or absence of 4-ANI, and cell death was assessed by propidium iodide uptake. (D) PARP immunoreactivity was examined in the spinal cord of IL-6–infused rats 4 days after initiation of IL-6 infusion. Magnification, ×20. (E) PARP activity was assessed in spinal cord tissue lysates generated from IL-6–infused rats, control rats, or rats both infused with IL-6 and given the PARP inhibitor 4-ANI intraperitoneally. Lysates were generated 4 days after initiation of IL-6 infusion. (F) IL-6 was infused into the spinal subarachnoid space of adult rats in the presence or absence of the PARP inhibitors 4-ANI or 3-AB given systemically. Hind limb grip strength was assessed daily for 7 days as described in Methods. *P < 0.05.

Article Snippet: Recombinant rat IL-6 (catalog no. 557008; BD Biosciences — Pharmingen) was made as a 2 μg/ml stock in 1 mg/ml BSA in PBS and used at final concentrations of 500 pg/ml and 2,000 pg/ml. iNOS inhibitor 1400W dihydrochloride (catalog no. ALX-270-073; Alexis Biochemicals) was made at 100 mM stock in water and used at a final concentration of 100 μM.

Techniques: Activation Assay, Activity Assay, Incubation, Marker, Generated

Journal: bioRxiv

Article Title: SOCS-1 inhibition of type I interferon limits Staphylococcus aureus skin host defense

doi: 10.1101/2020.09.28.317107

Figure Lengend Snippet: A) Gram staining of skin biopsies collected at day 1 post-MRSA skin infection from iKIR and scrambled KIR treated mice. Gram staining to label gram-positive bacteria is shown in purple/brown. Magnifications are as shown. Black arrows indicate extracellular MRSA clusters. Images are representative of 3–5 mice per group. B) Phagocytosis of GFP tagged MRSA by BMDM’s from SOCS1 fl and SOCS1 Δmyel mice or BMDM’s from WT mice treated with the SCR KIR or iKIR peptide. C) Determination of Bacterial killing of GFP tagged MRSA by BMDMs from B as described in the Materials and Methods. D) mRNA expression of Nos2 in the skin of infected mice treated with either SCR KIR or iKIR peptide at day 1 post-infection as determined via qPCR. E) Determination of bacterial killing of GFP-tagged MRSA as in C with BMDMs from WT mice treated with either SCR KIR, iKIR, or iKIR+ an iNOS inhibitor (1400W dihydrochloride). F) Measurement of nitric oxide in the supernatant of BMDMs from D . Data represent the mean ± SEM from 3–5 mice. *p < 0.05 vs. SOCS1 fl or SCR KIR treated mice. #p<.05 vs. iKIR treated mice.

Article Snippet: To determine the role of NO in microbial killing, cells were treated with 50 μM of the iNOS inhibitor 1400W dihydrochloride (Tocris).To measure the intensity of intracellular GFP fluorescence, extracellular fluorescence was quenched with 500 μg/mL trypan blue, and the GFP fluorescence was quantified using a fluorimeter plate reader.

Techniques: Staining, Infection, Bacteria, Expressing

Journal: International journal of molecular sciences

Article Title: Treatment of Status Epilepticus after Traumatic Brain Injury Using an Antiseizure Drug Combined with a Tissue Recovery Enhancer Revealed by Systems Biology.

doi: 10.3390/ijms241814049

Figure Lengend Snippet: Figure 2. Effect of in silico-identified drugs on tissue biomarkers of neuroprotection, oxidative stress, and neuroinflammation. (A) Neuronal viability. For a comparative viability analysis, neu- ronal survival in LPS/IFNγ+-treated neuronal–microglial co-cultures was set to 0% and that in co-cultures treated with iNOS inhibitor 1400 W (positive treatment control) to 100%. Trichostatin A at a 50 nM concentration increased neuronal survival from 0% to 73% (p < 0.001 compared with LPS/IFNγ+). The neuroprotective effect of TSA was almost as good as that of the positive control 1400 W (73% vs. 100%, p < 0.001). Also, chlorpromazine at a 50-µM concentration increased neuronal survival from 0% to 57% (p < 0.001) and calpain inhibitor at a 50-µM concentration increased neu- ronal survival from 0% to 47% (p < 0.001). Geldanamycin and tranylcypromine showed no consistent neuroprotection. IL10 had no effect on neuronal viability. Note that in untreated co-cultures without neuroinflammation (LPS/IFNγ−), neuronal viability was 38% of that in untreated BV2 neuronal cultures (p < 0.001) and 48% of that in 1400 W treated inflammation-exposed co-cultures (p < 0.001). Wells with cortical neurons only (BV2−cells without LPS/IFNγ+ exposure) showed the greatest viability. (B) Nitrite levels. For a comparative viability analysis, nitrite levels in LPS/IFNγ+-treated neuronal–microglial co-culture medium were set to 100% and that in co-culture medium treated with iNOS inhibitor 1400 W (positive treatment control) was set to 0%. Trichostatin A at a 50 nM concentration reduced the LPS/IFNγ+-induced nitrite release from 100% to 28% (p < 0.001). At a 10 nM concentration, there was a slight increase in nitrite levels to 121% (p < 0.01). Calpain inhibitor at 50 µM reduced nitrite release from 100% to −21% (p < 0.001). Chlorpromazine at 50 µM reduced nitrite levels to 81% (p < 0.001). At 10 µM, tranylcypromine reduced nitrite levels from 100% to 89% (p < 0.05) and at 1 µM, to 81% (p < 0.001). Interestingly, tranylcypromine at 100 µM had no effect (p > 0.05). Geldanamycin at 100 nM concentrations resulted in low nitrite levels, likely due to the cell death that was visualized in microscopic images (data not shown). IL10 had a mild effect on the LPS/IFNγ+-induced increase in nitrite levels, reducing it to 63% (p < 0.001). Note that neuron-only (BV2−) and LPS/IFNγ−neuronal–microglial co-cultures were not exposed to neuroinflammation, and thus showed no nitrite release to the culture medium. (C) TNFα levels. For a comparative viabil- ity analysis, TNFα levels in LPS/IFNγ+-treated neuronal–microglial co-culture medium were set to 100% and that in co-culture medium treated with TNFα inhibitor, IL10 (positive treatment control) to 0%. Trichostatin A at 10 nM concentration reduced TNFα levels from 100% to −2% (p < 0.001) and at 50 nM to −139% (p < 0.001), being better than the positive control. Calpain inhibitor at 50 µM reduced TNFα levels from 100% to −226% (p < 0.001). Chlorpromazine at 50 µM reduced TNFα levels from 100% to −21% (p < 0.001). The iNOS inhibitor, at 1400 W, reduced TNFα levels from 100% to 61% (p < 0.05). Abbreviations: BV2−, cortical neuronal cultures without BV2-microglial cells; IL10+, co-cultures treated with positive control anti-inflammatory incytokine, interleukin 10; LPS/IFNγ−, co-culture wells without lipopolysaccharides and interferon γ-induced neuroinflam- mation; LPS/IFNγ+, wells with lipopolysaccharides and interferon γ-induced neuroinflammation (untreated controls); TNFα, tumor necrosis factor α; 1400 W+, co-cultures treated with positive con- trol, inducible nitric oxide synthase (iNOS) inhibitor, 1400 W hydrochloride. Statistical significance: ***, p < 0.001; **, p < 0.01; *, p < 0.05 compared with LPS + IFNγ+ (linear regression in R). The number of independent experiments in panel A was three and in panels B-C was four. Data are presented as box plots with whiskers (minimum and maximum; box: interquartile range; line: median). Each dot shows an experimental value.

Article Snippet: Anti-inflammatory cytokine, interleukin 10 (IL10, 50 μg/mL, #500-M128, Peprotech, Rocky Hill, NJ, USA; final concentration of 50 ng/mL), and the neuroprotective inducible nitric oxide synthase (iNOS) inhibitor 1400 W hydrochloride (2 mM, #1415, Tocris, Bristol, UK; final concentration of 20 μM) were used as positive controls.

Techniques: In Silico, Control, Concentration Assay, Positive Control, Co-Culture Assay

Journal: The Journal of Clinical Investigation

Article Title: 3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

doi: 10.1172/JCI132987

Figure Lengend Snippet: ( A ) qPCR analysis of IL37 in ABCA12 WT and KO 3D models. n = 3. Data are represented as mean ± SD. NS, P = 0.068, unpaired t test. qPCR analysis of ( B ) SOCS1 and ( C ) SOCS3 in ABCA12 WT and KO 2D model cell lysates. Each dot represents the mean of 3 technical replicates. n = 3. Data are represented as mean ± SD. ** P ≤ 0.01, unpaired t test. ( D ) Representative immunoblot of p-STAT1 (Y701), total STAT1, and GAPDH proteins in untreated (–) or stimulated (+) with IFN-γ ABCA12 WT and KO cell lysates and ( E ) associated p-STAT1 quantitative analysis. n = 4. Data are represented as mean ± SD. * P ≤ 0.05, unpaired t test. The p-STAT1 blot was run in parallel, contemporaneously, with total STAT1 and GAPDH blots. ( F ) Representative NOS2 (green channel) and DAPI (blue channel) staining images of in vitro WT and HI 3D models, and ( G ) associated quantitative NOS2 analysis. Each dot represents the mean of relative NOS2 intensity from 3 independent images. Scale bars: 100 μm. n = 3. Data are represented as mean ± SD. * P ≤ 0.05, unpaired t test.

Article Snippet: Daily treatment used 1 μM of the selective and potent NOS2 inhibitor 1400W ( ) (1415, Tocris), 100 nM of the JAK1/3 inhibitor tofacitinib ( ) (sc-364726, Insight Biotechnology), or 50 μM of the NO-releasing agent S-Nitroso- N -acetyl-DL-penicillamine (SNAP) ( ) (sc-200319, Santa Cruz Biotechnology), which was added to the 3D models for 8 days.

Techniques: Western Blot, Staining, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: 3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

doi: 10.1172/JCI132987

Figure Lengend Snippet: Representative ( A ) IL-37, ( C ) IL-36α, ( E ) IL-36γ, ( G ) STAT1, ( I ) p-STAT1, ( J ) and NOS2 (green channel) and DAPI (blue channel) staining images of control skin and HI patient skin. Arrows indicate granular layer. Associated quantitative analysis of ( B ) IL-37, ( D ) IL-36α, ( F ) IL-36γ, ( H ) granular layer STAT1, and ( K ) NOS2 protein expression in control skin and HI patient skin. Each dot represents the mean of relative protein intensity from 3 independent images. n = 3 or 4. Data are represented as mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, unpaired t test. Scale bars: 100 μm.

Article Snippet: Daily treatment used 1 μM of the selective and potent NOS2 inhibitor 1400W ( ) (1415, Tocris), 100 nM of the JAK1/3 inhibitor tofacitinib ( ) (sc-364726, Insight Biotechnology), or 50 μM of the NO-releasing agent S-Nitroso- N -acetyl-DL-penicillamine (SNAP) ( ) (sc-200319, Santa Cruz Biotechnology), which was added to the 3D models for 8 days.

Techniques: Staining, Control, Expressing