Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a Naïve splenic CD4 + T cells from RORCE2 −/− or WT mice were stimulated under Th17-polarizing conditions for 5 days. The frequency of CD4 + RORγt + Th17 cells was measured by flow cytometry. b , c Summaries for the percentages of CD4 + RORγt + Th17 cells ( b ) and MFI of RORγt in Th17 cells ( c ) generated from RORCE2 −/− and WT mice on day 5 after in vitro polarization are shown. d Naïve splenic CD4 + T cells from RORCE2 −/− or WT mice were stimulated under Th17-polarizing conditions for 5 days. The frequency of CD4 + IL-17A + Th17 cells was measured by flow cytometry. e Summaries for the percentages of CD4 + IL-17A + Th17 cells generated from RORCE2 −/− and WT mice on day 5 after in vitro polarization are shown. f IL-17A production in culture supernatants was measured by ELISA. g , h Relative expressions of RORγt ( g ) and IL-17A ( h ) genes in Th17-polarized cells were measured by RT-qPCR. i – p Naïve splenic CD4 + T cells from RORCE2 −/− or WT mice were polarized under Th1- or Th2-polarizing conditions, and frequencies of Th1 ( i and j ) and Th2 ( m and n ) cells were analyzed by flow cytometry on day 6 after differentiation. Relative expression of the Tbx21 ( k ) or Gata3 ( o ) gene in Th1- or Th2-polarized cells was analyzed by RT-qPCR. The indicated cytokine production in cell culture supernatants was measured by ELISA ( l and p ). Mean ± SEM are shown, n = 5 independent experiments, unpaired two-tailed Student’s t -test ( b , c , e – h , j – l , n – p ). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Th1 and Th2 polarizations were performed using CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Flow Cytometry, Generated, In Vitro, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a The mean daily clinical scores for RORCE2 −/− and WT mice after EAE induction are shown (two-way analysis of variance (ANOVA), n = 14 biological independent animals). b Spinal cords collected on day 30 from indicated mice were stained with hematoxylin and eosin (HE) or luxol fast blue (LFB) to assess inflammation and myelin content, respectively. The outlines indicate inflammatory or demyelinated foci. Scale bars, 400 or 100 µm (magnified panels). c On day 30 after EAE induction, CD4 + T cells among leukocytes isolated from the spinal cord of indicated mice were gated and further analyzed to determine the frequencies of CD4 + IL-17A + Th17 cells. In addition, the absolute numbers of spinal cord-infiltrated CD4 + IL-17A + Th17 cells and total CD4 + T cells were also evaluated by flow cytometry. d , e On day 30 after EAE induction, CD4 + T cells among the leukocytes isolated from spinal cord of indicated mice were gated and further analyzed to determine the frequencies of CD4 + IFNγ + Th1 and CD4 + IL-4 + Th2 cells by flow cytometry. f Mononuclear cells were collected at day 8 from inguinal lymph nodes and further cultured ex vivo with MOG for 3 days. The concentrations of IL-17A, IFNγ, and IL-4 were measured by ELISA, respectively. Mean ± SEM are shown, n = 5 biological independent animals, unpaired two-tailed Student’s t -test ( c – f ). Experimental mice were between 10 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Th1 and Th2 polarizations were performed using CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Staining, Isolation, Flow Cytometry, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a Map of the investigated interactions. Since we found NlaIII to be the only restriction enzyme that allowed sufficiently high-resolution analysis of RORCE, NlaIII was finally used to digest the RORCE region. The positions of NlaIII sites (vertical lines) in RORCE used in this 3C analysis are indicated as test sites 1 to 8 (TS1 to TS8). The NlaIII site in RORγt gene promoter was taken as the anchor site (AS), from which possible interactions with other regions of RORCE were quantified. The primers used for 3C-qPCR are indicated by simple black arrows. The negative control site (NCS) is another NlaIII site upstream of RORCE, which does not interact with the AS. b – f Quantitative analysis of a 3C-qPCR assay assessing the RORCE region. The data represent relative interaction frequencies between AS and the other tested sites (TS1 to TS8) in the RORCE region. Relative interaction frequencies were determined by qPCR relative to standard curves as described in the “Methods” section. Data points represent the mean ± SEM for PI-stimulated EL4 ( b , n = 4 independent experiments), FACS-purified CD4 + IL17 + Th17 ( c , n = 5 independent experiments), Th17-polarized ( d , n = 5 independent experiments), Th1-polarized ( e , n = 5 independent experiments), and Th2-polarized cells ( f , n = 5 independent experiments) from WT mice. Th17 D, Th1 D, and Th2 D represent Th17, Th1, or Th2 differentiation under the appropriate polarization conditions, respectively, and N represents naïve cells. All P -values are based on unpaired two-tailed Student’s t -test ( b – d ). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Th1 and Th2 polarizations were performed using CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Negative Control, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a The ranking of predicted transcriptional factors (TFs) capable of binding to RORCE was performed based on relative scores predicted with the JASPAR database ( http://jaspar.genereg.net ). b Red vertical line shows SOX-5 binding site (SOX-5-BS) in RORCE, which is very close to TS3. c , d A ChIP-qPCR assay was performed to determine the enrichment of SOX-5 in SOX-5-BS and RORγt promoter (RORγt p) in PI-stimulated EL4 cells ( c ) and WT Th17-polarized cells ( d ). e – h A ChIP-loop assay with an anti-SOX-5 antibody or control mouse IgG was performed to quantify relative interaction efficiency between the AS and TS3 mediated by SOX-5 in PI-stimulated EL4 ( e ), Th17-polarized ( f ), Th1-polarized ( g ), and Th2-polarized cells ( h ) from WT mice. i The sequence of SOX-5-BS (AACAA) in RORCE was deleted by using CRISPR/Cas9 technology (top). The knockout allele was confirmed by sequencing (bottom). j A ChIP-qPCR assay was performed to test the enrichment of SOX-5 in SOX-5-BS in Th17-polarized cells from SOX-5-BS-deficient (SOX-5-BS −/− ) or WT mice. k , l Quantitative analysis of 3C-qPCR ( k ) and ChIP-loop assays ( l ) evaluating the interaction between TS3 and AS in Th17-polarized cells from SOX-5-BS-deficient or WT mice was performed. Th17 D, Th1 D, and Th2 D represent Th17, Th1, or Th2 differentiation under the corresponding polarization conditions, respectively, and N represents naïve cells. Mean ± SEM are shown, n = 4 independent experiments, unpaired two-tailed Student’s t -test ( b – h , j – l ). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Th1 and Th2 polarizations were performed using CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Binding Assay, ChIP-qPCR, Control, Sequencing, CRISPR, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha D23 blocks T helper 2 cell polarization via IL-10 – mediated interference with dendritic cell activation

doi: 10.1101/2020.06.30.175414

Figure Lengend Snippet: (a-f) IL-5 and IL-13 supernatant levels are reduced with AOB pretreatment of PBMC prior to Th2 differentiation cocktail stimulation; measured by ELISA of supernatants 72h post-stimulation. IL-4 gene expression is also reduced with AOB pretreatment; measured by qPCR 72h post-stimulation. AOB pre-treatment prior to Th2 differentiation cocktail stimulation of PBMCs from donor A reduced the levels of IL-5 (a) & IL-13 (b) as well as IL-4 (c) gene expression (n ≥ 12, one-way ANOVA with multiple comparisons or unpaired t-test). (d-f) IL-5 (d), IL-13 (e), and IL-4 (f) levels/expression are reduced in AOB-pretreated PBMC from 3-5 different donors (n ≥ 6 replicates per donor, shown on the figure is unpaired t-test p value for individual donors, unpaired t-test p value for aggregated donors: p<0.0001 for IL-5, p<0.01 for IL-13, p<0.001 for IL-4). (g, h) The number of CD4+ IL-5+ cells is reduced with AOB pretreatment of PBMC following Th2 differentiation cocktail stimulation; measured by flow cytometry with anti-CD4 PerCP cy5.5 and anti-IL-5 PE antibodies applied 72 hours post-stimulation. (g) Representative dot plot from donor C cells showing the reduction in CD4+ and IL-5+ cells. (h) The percentage of Th2 cells as CD4+ IL-5+ are reduced with AOB pretreatment (n=3, donor C, unpaired t-test). IL-5 (i) and IL-13 (j) supernatant levels are reduced in the presence of AOB 7 days post-stimulation with SEB (1 μg/mL), measured by ELISA (n=3 per donor, 3 donors, unpaired t-test, p<0.0001 for IL-5 and IL-13 for aggregated donor data sets).

Article Snippet: PBMC were then stimulated using either the CellXvivo human Th2 differentiation kit (R&D systems) following the manufacturer’s recommendations, or 1μg/mL SEB (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha D23 blocks T helper 2 cell polarization via IL-10 – mediated interference with dendritic cell activation

doi: 10.1101/2020.06.30.175414

Figure Lengend Snippet: (a-d) IFNγ (a, b) and IL-12p70 (c, d) supernatant levels are increased with AOB pretreatment of PBMC prior to Th2 kit stimulation; measured by ELISA. IFNγ (a) and IL-12p70 (c) levels are increased in PBMC from donor A (n=9, one-way ANOVA with multiple comparisons). IFNγ (b) is induced in AOB pre-treated PBMC from 5 donors and IL-12p70 (d) is induced in PBMC from 2 of 3 donors tested (n ≥ 6 per donor, unpaired t-test, p<0.0001 for IFNγ and p<0.0015 for IL-12 aggregated donor datasets). (e, f) The number of CD4+ IFNγ+ cells is increased with AOB pretreatment of PBMC prior to Th2 differentiation cocktail stimulation as measured by staining with anti-CD4 PerCP cy5.5 and anti-IFNγ FITC antibodies 72 hours post-stimulation. (e) Representative dot plot from donor C PBMC showing an increase in the percentage of CD4+ and IFNγ+ cells. (f) Percentage of Th1 cells as CD4+ IFNγ+ are increased with AOB pretreatment (n=6, unpaired t-test). (g, h) Addition of IFNγ or IL-12 neutralizing antibodies to PBMC prior to AOB pretreatment does not prevent AOB inhibition of Th2. IFNγ (g) production is reduced in presence of 10μg/mL IFNγ or IL-12 neutralizing antibodies in PBMC from 3 donors (n=3 per donor, one point per donor, one-way ANOVA with multiple comparisons). AOB-mediated reduction in IL-5 production (h) is unaffected in presence of 10μg/mL IFNγ or IL-12 neutralizing antibodies in PBMC from 3 donors (n=3 per donor, one point per donor, one-way ANOVA with multiple comparisons).

Article Snippet: PBMC were then stimulated using either the CellXvivo human Th2 differentiation kit (R&D systems) following the manufacturer’s recommendations, or 1μg/mL SEB (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Inhibition

Journal: bioRxiv

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha D23 blocks T helper 2 cell polarization via IL-10 – mediated interference with dendritic cell activation

doi: 10.1101/2020.06.30.175414

Figure Lengend Snippet: (a-d) Expression of MHC II (a-b) or CD86 (c-d) in CD11c+ cells is reduced in PBMC treated with AOB prior to Th2 differentiation cocktail stimulation; measured by flow cytometry in Th2-stimulated PBMC in the presence or absence of AOB. (a, c) Representative fluorescence intensity plots of anti-MHC II FITC (a) or anti-CD86 PE (c) stained PBMC from donor C in the presence (red) or absence (grey) of AOB. (b, d) Relative MHC II (b) or CD86 (d) expression in CD11c+ positive cells is reduced with AOB treatment in 3 donors (n=3 per donor, unpaired t-test, p<0.0001 for MHC II and CD86 aggregated donor datasets). (e) IL-10 production is induced by AOB in PBMC from 3 donors 24h post-stimulation; measured by ELISA of culture supernatants (n=3 per donor, p=0.1095 for aggregated donor data set). (f-h) Addition of IL-10 neutralizing antibodies to PBMC prior to AOB pretreatment interfered with AOB’s inhibition of Th2. IL-10 (f) production by Th2-stimulated PBMC was reduced with the addition of IL-10 neutralizing antibody but not with isotype control in 3 donors (n=3 per donor, one point per donor, one-way ANOVA with multiple comparisons). AOB-mediated fold reduction in IL-5 (g) was obstructed by the addition of IL-10 neutralizing antibody compared to isotype control in 2 of 3 donors tested (n=3 per donor, unpaired t test). AOB-mediated reduction in CD86 expression (h) in CD11c+ cells is hindered with the addition of IL-10 neutralizing antibody but not with isotype control (n=3, donor D, one-way ANOVA with multiple comparisons).

Article Snippet: PBMC were then stimulated using either the CellXvivo human Th2 differentiation kit (R&D systems) following the manufacturer’s recommendations, or 1μg/mL SEB (Sigma).

Techniques: Expressing, Flow Cytometry, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Inhibition, Control

Journal: bioRxiv

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha D23 blocks T helper 2 cell polarization via IL-10 – mediated interference with dendritic cell activation

doi: 10.1101/2020.06.30.175414

Figure Lengend Snippet: (a, b) AOB metabolites nitrite (a) and nitric oxide (b) are produced by live but not heat killed AOB after one hour incubation in AOB culture media (n=3, unpaired t-test). (c, d) Fold-reduction in IL-5 (c) and IL-13 (d) mediated by AOB (red) is not significantly different from fold-reduction mediated by heat killed AOB (blue); measured by ELISA in cell culture supernatant collected 72h post-stimulation with the Th2 stimulation cocktail (n ≥ 6 per donor, 5 donors, one datapoint per donor, unpaired T test). (e, f) Fold reduction in MHC II (e) and CD86 (f) expression in CD11c+ cells mediated by live AOB (red) is not significantly different from fold-reduction mediated by heat killed AOB (blue); measured by flow cytometry 72h after Th2 stimulation in donor C (n=6, unpaired T test). (g, h) IFNγ (g) and IL-12p70 (h) production by PBMC are not as strongly induced by heat killed AOB (blue) as by live AOB (red); measured by ELISA from supernatant of PBMC culture collected 72h post-stimulation (n ≥ 3 per donor, 3 or 5 donors, one datapoint per donor, one-way ANOVA with multiple comparisons).

Article Snippet: PBMC were then stimulated using either the CellXvivo human Th2 differentiation kit (R&D systems) following the manufacturer’s recommendations, or 1μg/mL SEB (Sigma).

Techniques: Produced, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Flow Cytometry