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