Journal: JCI Insight

Article Title: Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice

doi: 10.1172/jci.insight.131277

Figure Lengend Snippet: IG alcohol–fed WT mice (male C57BL/6, 12 weeks old, n = 10–12/group, 3 independent experiments) were treated with RORγt inhibitor (50 mg/kg/weight vs. vehicle, oral gavage 4 times per week, or vehicle) or anti–IL-17A Ab (25 mg/kg/weight vs. IgG, i.p., 1 time per week). (A) Serum levels of IL-17A (pg/mL), (B) ALT (IU/L), and blood EtOH (nM) were measured. (C) Livers were stained with H&E and Sirius Red, positive area was calculated as percentage, and micrographs are shown using ×20 objective. (D) Expression of fibrogenic and (E) inflammatory gene mRNA. Data are shown as fold change (vs. IgG-treated mice). One-way ordinary ANOVA for multiple comparisons was applied. *P < 0.05; **P < 0.01 (see Supplemental Figures 4–6).

Article Snippet: We thank K. Diggle for her excellent technical support, G. Arteel (University of Louisville Health Sciences Center) for providing lung cDNA from IG alcohol– and pair-fed mice, J. Zapf (Visionary Pharmaceuticals) for providing RORγt inhibitor, M. Elloso (Janssen) for providing anti–IL-17A Ab, and Amgen for providing IL-17ra –/– mice.

Techniques: Staining, Expressing

Journal: JCI Insight

Article Title: Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice

doi: 10.1172/jci.insight.131277

Figure Lengend Snippet: Brains and cerebrospinal fluid (CSF) were collected from IG alcohol–fed WT mice with or without RORγt inhibitor or with or without anti–IL-17A Ab (male C57BL/6, 12 weeks old, n = 12/group, 3 independent experiments). (A) Coronal brain sections were immunostained for induction of brown adipocytes 1 (Iba1) (hippocampus, ×20 and ×100 objectives) or glial fibrillary acidic protein (GFAP) (cortex, ×4 and ×40 objectives). (B) Positive area was calculated as percentage. (C) Expression of Iba1 and GFAP mRNA. Data are shown as fold change. (D) CSF levels of IL-17A and TNF-α were measured by ELISA (pg/mL). (E) Expression of TNF-α mRNA was measured. Two-tailed Student’s t test and 1-way ordinary ANOVA for multiple comparisons were applied. *P < 0.05 (see Supplemental Figures 7–10).

Article Snippet: We thank K. Diggle for her excellent technical support, G. Arteel (University of Louisville Health Sciences Center) for providing lung cDNA from IG alcohol– and pair-fed mice, J. Zapf (Visionary Pharmaceuticals) for providing RORγt inhibitor, M. Elloso (Janssen) for providing anti–IL-17A Ab, and Amgen for providing IL-17ra –/– mice.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: JCI Insight

Article Title: Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice

doi: 10.1172/jci.insight.131277

Figure Lengend Snippet: (A) Serum levels of IL-17A cytokine were measured in excessive drinkers with normal ALT (<40 U/L, n = 21) or elevated ALT (≥40 U/L, n = 36) and healthy individuals (n = 18). Data are shown as IL-17A concentration, pg/mL, presented as mean ± SD. Two-way ordinary ANOVA. Patient information is summarized in Supplemental Tables 1 and 2. (B) Naive mice (male C57BL/6J, n = 4–6/experimental condition) were subjected to 2BC sessions to determine baseline voluntary ethanol drinking. Mice were then exposed to either Air (nondependent mice) or CIE (dependent mice). CIE produced an escalation of voluntary ethanol drinking during 2BC sessions conducted 3 to 7 days into withdrawal from vapor (vs. nondependent mice). Dependent and nondependent mice (n = 4–6/group) were treated with or without anti–IL-17A Ab, or with or without RORγt inhibitor, and (C) serum IL-17A was measured (after CIE or Air, withdrawal day 4, and 24 hours after 2BC-CIE; also see Supplemental Figure 11A). *P < 0.05.

Article Snippet: We thank K. Diggle for her excellent technical support, G. Arteel (University of Louisville Health Sciences Center) for providing lung cDNA from IG alcohol– and pair-fed mice, J. Zapf (Visionary Pharmaceuticals) for providing RORγt inhibitor, M. Elloso (Janssen) for providing anti–IL-17A Ab, and Amgen for providing IL-17ra –/– mice.

Techniques: Concentration Assay, Produced

Journal: JCI Insight

Article Title: Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice

doi: 10.1172/jci.insight.131277

Figure Lengend Snippet: (A and B) Voluntary alcohol intake (g ethanol/kg body weight) was measured during 2BC sessions (2 hours) in Air and CIE mice with or without anti–IL-17A Ab (or with or without RORγt inhibitor). (A) Multiple doses of anti–IL-17A Ab were administered on consecutive days (the regimen is shown, red arrows). The most significant effect was detected on the first day of treatment (withdrawal day 4: effect of Ab, F[1,19] = 6.6, and P < 0.05; effect of CIE, F[1,19] = 9.7, and P < 0.01; Ab by CIE interaction, F[1,19] = 5.1, and P < 0.05) and persisted throughout the week (withdrawal days 4–7: effect of Ab, F[1,19] = 3.5, and P < 0.08; see Supplemental Figure 10). (B) Air and CIE mice with or without RORγt inhibitor. There was no immediate effect of RORγt inhibitor on voluntary ethanol drinking. The mice were then exposed to an additional week of CIE (or Air) and resumed 2BC sessions. The most significant effect was observed on withdrawal day 3 (effect of inhibitor, F[1,20] = 10.1, and P < 0.01; effect of CIE, F[1,20] = 47.3, and P < 0.001; inhibitor by CIE interaction, F[1,20] = 7.2, and P < 0.05) but persisted throughout the week (withdrawal days 3–6: effect of inhibitor, F[1,20] = 5.5, and P < 0.05). The regimen of IL-17A inhibition is specified (red arrows). One-way ordinary ANOVA for multiple comparison was applied.

Article Snippet: We thank K. Diggle for her excellent technical support, G. Arteel (University of Louisville Health Sciences Center) for providing lung cDNA from IG alcohol– and pair-fed mice, J. Zapf (Visionary Pharmaceuticals) for providing RORγt inhibitor, M. Elloso (Janssen) for providing anti–IL-17A Ab, and Amgen for providing IL-17ra –/– mice.

Techniques: Inhibition, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted ROR γ t –/– CD4 – CD8 – or WT CD4 – CD8 – in the presence of different concentrations of IMU-935 ( A ) ( n = 2–4/group, from 4 experiments) or RORγt modulators ( B ) (1 μM; n = 4/group, from 4 experiments). Percentages of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + thymocytes are shown. ( C and D ) Percentages of live cells among CD4 + CD8 + thymocytes relative to the vehicle control different times after culture in the presence of indicated concentrations of IMU-935 ( C ) or RORγt modulators ( D ) (1 μM; n = 3/group, from 1 experiment). Thymocytes from ROR γ t –/– mice were used as a control. ( E ) Picture and cellularity of the thymus from mice ( n = 8–9/group; n = 8 for vehicle and IMU-935 groups, and n = 9 for Cpd-1 and cintirorgon groups) treated with vehicle or indicated RORγt inhibitors or cintirorgon (100 mg/kg orally, twice daily) for 28 days. ( F ) H&E staining (scale bar: 500 μm) of the thymus from mice treated as described in E . M, medullar; C, cortex. Bottom: The ratio of the medullary/cortical region. ( G ) Flow cytometric analysis of CD4 and CD8 in the thymocytes from mice treated as described in E . ( H ) TUNEL staining (top; scale bar: 500 μm) and percentage of TUNEL-positive apoptotic cells in thymus obtained from mouse treated as described in E . Bottom right: The qRT-PCR analysis of Bcl2l1 mRNA. ( I ) Number of alive, human CD4 + CD8 + thymocytes cultured in the presence of indicated RORγt inhibitors (1 μM) for 24 hours (thymocytes collected from 1 donor, n = 3 replicates/group). Data were assessed by 1-way ANOVA with Dunnett’s post hoc test ( A – H ) or 2-tailed Student’s t test ( I ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Ex Vivo, Control, Staining, TUNEL Assay, Quantitative RT-PCR, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) Venn diagram displaying the number of DEGs (1.5-fold, up or down; P < 0.05) identified by RNA-Seq assays in WT versus ROR γ t –/– thymocytes and in WT thymocytes treated with IMU-935 (1 μM) or not. ( B and C ) Volcano plots displaying the global gene expression. The horizontal dotted line marks P = 0.05, the vertical dotted lines mark fold change of ±1.5. ( D ) Heatmap showing antiapoptotic, apoptotic, and cell cycle gene expression in ROR γ t –/– or IMU-935–nontreated (WT-vehicle control) or treated (WT–IMU-935) thymocytes. Gene expression was normalized by total counts of each gene. ( E ) Heatmap depicting changes in the activity of upstream regulators predicted by IPA. Activation z score indicates increased (orange; z > 0) or decreased (blue; z < 0) activity. All z score values are labeled within the corresponding cell. Genes shown are known to regulate apoptosis ( Mycn , E2f1 , Esr1 ), survival ( Nfkbia , TP53 ), cell cycle ( Gli1 , Cdkn2a ), oxidative stress ( Nfe2l2 ), and thymocyte development ( Ctnnb1 , Lef1 ). ( F ) Heatmap showing the activity of the pathways determined by IPA. Pathway activity was obtained by comparing WT and ROR γ t –/– thymocytes (left column) and IMU-935–nontreated versus -treated WT thymocytes (right column). N/A indicates pathway status is not predictable due to the small number of genes affected by IMU-935 treatment in thymocyte RNA-Seq. ( G ) Genome-wide RORγt-DNA binding signal intensity determined by ChIP-Seq assays near the TSS in ROR γ t –/– or WT (RORγt + ) or WT–IMU-935 thymocytes. ( H ) RORγt binding peaks at the Bcl2l1 locus. The results are the representative of overlays from 2 separate experiments. Veh, vehicle.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: RNA Sequencing, Gene Expression, Control, Activity Assay, Activation Assay, Labeling, Genome Wide, Binding Assay, ChIP-sequencing

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) Changes in Th17 differentiation after knockout of indicated genes, relative to nontargeting group (NonT; 100%) in Cas9-expressing CD4 + T cells ( n = 3–10/group from 3 pooled independent experiments). ( B ) Relative luciferase activity from a promoterless control (pGL3) or RORγt reporter (RORBE) in HEK293T cells transfected with indicated expression plasmids for 24 hours. ( C ) A visualization of the computer-predicted interaction between RORγt and CBFβ (top left) or RUNX1 and CBFβ (top right). Dashed circle highlights the interaction interface between indicated proteins. The cyan fragment indicates the RORγt amino acids 403–413 critical for binding to CBFβ. The black box in the bottom panel indicates the amino acids with >40% contact frequency with CBFβ; the white box indicates <40% contact frequency. ( D and E ) IP analysis of the RORγt-CBFβ interaction in ROR γ t –/– CD4 + T cells expressing GFP (EV) or with Flag-RORγt polarized in Th17 conditions ( D ) in ROR γ t –/– or WT thymocytes ( E ) and treated with indicated concentrations of IMU-935. IP with anti–Flag-RORγt ( D ) or anti-RORγt ( E ) antibody and immunoblot with anti-CBFβ antibody. Input was analyzed by Western blot (bottom 2 panels). Right: The relative intensity of immunoprecipitated CBFβ band. ( F ) Flow cytometric analysis of RORγt (top), RUNX1 (middle), and CBFβ (bottom) levels in differentiated Th17 cells, peripheral naive CD4 + T cells, and different subsets of thymocytes. ( G and H ) IP analysis of the RORγt-CBFβ interaction in control (NonT) or Runx1 -deleted Th17 cells (sg Runx1 ) ( G ) or thymocytes expressing EV or RUNX1 ( H ) treated with indicated concentrations of IMU-935, similar to what is described in D and E . Data represent 2 ( F ), 3 ( H ), or 4 ( B , D , E , and G ) independent experiments. Data were assessed by 1-way ANOVA with Dunnett’s ( A , D , and E ), Tukey’s ( B ) post hoc test, or 2-tailed Student’s t test ( G and H ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Knock-Out, Expressing, Luciferase, Activity Assay, Control, Transfection, Binding Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A – C ) IP analysis of the RORγt-CBFβ interaction in HEK293T cells with indicated expression plasmids for WT ( A ), deletion mutation ( B ), or alanine scanning mutation ( C ) RORγt. ΔDBD, DNA binding domain-deleted RORγt; ΔHinge, hinge-deleted RORγt; ΔLBD, Lihand-binding domain-deleted RORγt. ( D ) The percentage of Th17 differentiation activity supported by the RORγt with the indicated point mutation expressed in ROR γ t –/– CD4 + T cells relative to those expressing WT RORγt (defined as 100%). ROR γ t –/– CD4 + T cells expressing GFP alone (EV) or with WT RORγt or RORγt with the indicated single amino acid mutated to alanine were polarized under Th17 conditions. ( E ) Visualization of computer-predicted interaction between RORγt and CBFβ. The cyan fragment indicates RORγt amino acids 403–413. R407, L410, and E412 of RORγt critical for binding to CBFβ are indicated. Red dashed lines indicate the ionic lock between R407 and E336. ( F ) IP analysis of CBFγ interaction with RORβt or R407A/L410A DM. ( G and H ) Relative luciferase activity from HEK293T cells transfected with pGL3 control or RORγt reporter (RORBE) together with plasmids expressing WT or RORγt with indicated mutation ( G ) with or without CBFβ ( H ). ( I ) The percentage of Th17 differentiation activity supported by the RORγt with indicated mutation expressed in ROR γ t –/– CD4 + T cells relative to those expressing WT RORγt (defined as 100%). ( J ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted ROR γ t –/– CD4 – CD8 – transduced with WT or RORγt with indicated mutation. Right: The summary of the percentages of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + cells. Data represent 3 ( D and G ) or 4 ( H – J ) independent experiments; representative Western blots are shown from 3 independent experiments ( A – C , and F ). Data were analyzed by 1-way ANOVA with Dunnett’s ( D ) or Tukey’s ( G – J ) post hoc test. * P < 0.05; ** P < 0.01. WCL, whole-cell lysate.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Luciferase, Transfection, Control, Ex Vivo, Transduction, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) IP analysis of the RORγt-CBFβ interaction in Th17 cells (left) or thymocytes (right) from indicated mice. Cell lysates from differentiated Th17 cells and thymocytes were subjected to IP with anti-RORγt antibody and immunoblotting with anti-CBFβ or (top) and anti-RORγt (bottom) antibody. Input CBFβ and RORγt were analyzed by Western blot (bottom 2 panels). Representative Western blots are shown from 3 independent experiments. ( B ) Flow cytometric analysis and percentage of IL-17A + cells among CD4 + T cells from indicated mice polarized under Th17 conditions for 60 hours ( n = 4 mice/group, from 1 experiment). ( C ) Clinical score of EAE among indicated mice ( n = 6/group) different days after induction of disease by immunization with MOG 35–55 . ( D ) Flow cytometric analysis (left 2 panels), percentages (third panel), and numbers (right) of CD4 + T cells infiltrated into the CNS of EAE-induced mice shown in C . ( E ) Flow cytometric analysis (left 2 panels), percentages (third panel), and numbers (right) of CNS-infiltrated CD4 + T cells producing IL-17A in EAE-induced mice shown in C . ( F ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted WT or ROR γ t DM/DM CD4 – CD8 – cells. Right: Summary of the percentage of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + cells ( n = 3 mice/group, from 1 experiment). ( G ) Percentage of live cells among thymocytes from indicated mice and cultured for different times in vitro ( n = 4 mice/group, from 1 experiment). Data were analyzed by 2-tailed Student’s t test ( B – G ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Western Blot, Ex Vivo, Cell Culture, In Vitro