flowx human th2 cell multi color flow cytometry kit  (R&D Systems)

Journal: Scientific Reports

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha blocks T helper 2 cell polarization via the anti-inflammatory cytokine IL-10

doi: 10.1038/s41598-021-93299-1

Figure Lengend Snippet: AOB inhibit Th2 immune polarization in human PBMC. ( 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 72 h post-stimulation. IL-4 gene expression is also reduced with AOB pretreatment; measured by qPCR 72 h post-stimulation. AOB pre-treatment prior to Th2 differentiation cocktail stimulation of PBMCs from donor A reduced the levels of IL-5 ( a ) and 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 to 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 h 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 1 μg/mL SEB (Sigma).

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

Journal: Scientific Reports

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha blocks T helper 2 cell polarization via the anti-inflammatory cytokine IL-10

doi: 10.1038/s41598-021-93299-1

Figure Lengend Snippet: AOB induce Th1 polarization but Th1 are not necessary for Th2 inhibition. ( 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 h 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 10 µg/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 10 µg/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 1 μg/mL SEB (Sigma).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha blocks T helper 2 cell polarization via the anti-inflammatory cytokine IL-10

doi: 10.1038/s41598-021-93299-1

Figure Lengend Snippet: AOB-mediated Th2 inhibition requires IL-10 and is associated with a suppression of dendritic cell activity. ( 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 24 h 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 1 μg/mL SEB (Sigma).

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

Journal: Scientific Reports

Article Title: The ammonia oxidizing bacterium Nitrosomonas eutropha blocks T helper 2 cell polarization via the anti-inflammatory cytokine IL-10

doi: 10.1038/s41598-021-93299-1

Figure Lengend Snippet: Structural components could be responsible for AOB-mediated Th2 inhibition. ( a , b ) AOB metabolites nitrite ( a ) and nitric oxide ( b ) are produced by live but not heat killed AOB after 1 h 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 72 h 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 72 h 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 72 h 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 1 μg/mL SEB (Sigma).

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