11 research outputs found
Impaired IgG and GC responses in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice.
<p>(<b>A</b>) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup>, and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. (<b>B</b>) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR<i>lpr</i> mice were used as negative and positive controls, respectively. (<b>C</b>) Impaired IgG responses to a TD Ag. WT (<i>n</i> = 3), CD22<sup>−/−</sup> (<i>n</i> = 4), CD154<sup>TG</sup> (<i>n</i> = 6), and CD154<sup>TG</sup>CD22<sup>−/−</sup> (<i>n</i> = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220<sup>+</sup> B cells (red) and GC GL7<sup>+</sup>B220<sup>+</sup> B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22<sup>−/−</sup> mice, and detectable GL7<sup>+</sup>B220<sup>+</sup> B cells within the follicles of CD154<sup>TG</sup> mice, but not in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p<0.05, ††p<0.01). (<b>D</b>) Reduced GC B cells in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice as quantified by flow cytometry analysis. Mice were immunized with NP-CGG in alum, with spleens analyzed for GL7<sup>+</sup>B220<sup>+</sup> B cells on day 10. Contour plots show mean (±SEM) GL7<sup>+</sup> cell frequencies among total B220<sup>+</sup> cells from <i>n</i> = 3 mice of each genotype. Bar graphs show mean (±SEM) GL7<sup>+</sup> B cell numbers from naive (open bars) and immunized (filled bars) mice. In the contour plots, mean B10 cell frequencies significantly lower than those of WT mice are indicated by asterisks (**p≤0.01). In the bar graphs, means significantly different between naïve and immunized mice of the same genotype are indicated by asterisks (*p≤0.05); crosses for CD154<sup>TG</sup>CD22<sup>−/−</sup> mice indicate that mean GL7<sup>+</sup> cell numbers were significantly reduced relative to all other genotypes (††p<0.01).</p
B cell development in CD154<sup>TG</sup>CD22<sup>−/−</sup> and CD154<sup>TG</sup> mice.
<p>(<b>A</b>) B cell CD154 expression in WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup> and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice. Blood and spleen B220<sup>+</sup> B cells were assessed for CD154 expression by immunofluorescence staining with flow cytometry analysis. Dashed lines delineate borders between CD154<sup>+</sup> and CD154<sup>−</sup> cells as determined using WT lymphocytes. (<b>B</b>) Ectopic CD154 expression is B cell-restricted in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice. CD154 expression by blood and spleen B220<sup>+</sup> and B220<sup>−</sup> mononuclear cells was assessed by immunofluorescence staining with flow cytometry analysis. Results represent those obtained in 6 pairs of mice. (<b>C</b>) Increased CD5 expression by B cells from CD22<sup>−/−</sup> and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice. Spleen B cells were assessed for CD5 expression by immunofluorescence staining. Gates indicate percentages of CD5<sup>+</sup> B cells among total B220<sup>+</sup> cells. (<b>D</b>) Spleen CD1d<sup>hi</sup>B220<sup>+</sup> B cell localization. Tissue sections from WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup>, and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice were stained with B220 (FITC, green) and CD1d (PE, red) mAbs. Merged images highlight CD1d<sup>hi</sup>B220<sup>+</sup> cells (yellow). (A–C) Results are representative of ≥3 mice of each genotype. (<b>E</b>) Spleen CD1d<sup>hi</sup>B220<sup>+</sup> B cell numbers in WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup> and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice as determined by immunofluorescence staining. Results represent the mean (±SEM) from ≥3 mice of each genotype. (<b>F</b>) CD154<sup>TG</sup>CD22<sup>−/−</sup> B cells are hyper-responsive to CD40 signals. Spleen B cells from WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup>, and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice were cultured with mitogenic CD40 mAb or anti-IgM Ab for 72 h, with [<sup>3</sup>H]-thymidine incorporation assessed during the final 18 h of culture. Values represent means (±SEM) of triplicate cultures. (E–F) Means significantly different from WT values are indicated by asterisks (**p<0.01), and between other genotypes by crosses (††p<0.01). (<b>G</b>) CD154<sup>TG</sup>CD22<sup>−/−</sup> B cell proliferation in response to LPS stimulation. Cell division of CFSE-labeled B cells from WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup> and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice following LPS stimulation was quantified after 72 h in culture by flow cytometry. (F–G) Results are representative of 2 independent experiments with similar results. (<b>H</b>) Enhanced survival by CFSE-labeled B cells from CD154<sup>TG</sup> and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice after 10 days in culture without mitogenic stimulation as assessed by flow cytometry. Results represent 3 independent experiments producing similar results.</p
Spleen B10 and B10pro cells expand dramatically in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice.
<p>(<b>A</b>) Blood, spleen, inguinal LN, and peritoneal cavity B10 cell numbers in WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup>, and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice. Leukocytes from the indicated tissues were cultured with LPS, PMA, and ionomycin for 5 h to induce IL-10 production, with monensin included in the cultures to block IL-10 secretion (L+PIM stimulation). The cells were then stained for cell surface CD19 and cytoplasmic IL-10 with flow cytometry analysis. Monensin treated cells served as negative controls for IL-10 expression. Representative contour plots gated on CD19<sup>+</sup> cells are shown with the percentage of IL-10<sup>+</sup> cells indicated. Bar graphs represent mean (±SEM) B10 cell frequencies (%) and numbers (#) from ≥3 mice of each genotype. (<b>B</b>) Similar B10 cell surface phenotypes among mouse genotypes. Splenocytes were cultured with L+PIM for 5 h and then stained for cell surface molecules and cytoplasmic IL-10. Histograms indicate cell surface molecule expression by IL-10<sup>+</sup> (thick lines) and IL-10<sup>−</sup> (thin lines) B cells. Dashed histograms represent isotype-matched control mAb staining. Results are representative of ≥3 mice of each genotype analyzed. (<b>C</b>) Spleen B10+B10pro cell frequencies and numbers. Splenocytes were cultured with an agonistic CD40 mAb, with L+PIM added during the final 5 h of 48 h cultures. Cytoplasmic IL-10<sup>+</sup> B cells were identified as in (A). Bar graphs show means (±SEM) from ≥3 mice of each genotype. (A,C) Significant differences from WT mice are indicated: *p<0.05, **p<0.01. Means significantly different between CD154<sup>TG</sup>CD22<sup>−/−</sup> and CD154<sup>TG</sup> mice are indicated: †p<0.05, ††p<0.01.</p
Endogenous B10 cells regulate IgG isotype switching in normal mice.
<p>(<b>A–B</b>) CD22 mAb treatment preferentially depletes CD1d<sup>hi</sup>CD5<sup>+</sup> and B10 cells. Seven days after WT mice were given CD22 or control mAb, splenocytes were isolated and cultured in the presence of L+PIM for 5 h followed by cell surface CD19, CD1d and CD5, and intracellular IL-10 staining, with flow cytometry analysis. IL-10<sup>−/−</sup> mice given control mAb were also evaluated for comparison. (A) CD22 mAb treatment preferentially depletes CD1d<sup>hi</sup>CD5<sup>+</sup> B cells. Representative CD1d<sup>hi</sup>CD5<sup>+</sup> B cell frequencies are indicated. (B) CD22 mAb treatment preferentially depletes B10 cells. Representative cytoplasmic IL-10<sup>+</sup> B cell frequencies among spleen CD19<sup>+</sup> cells are indicated. Bar graphs show mean (±SEM) frequencies and numbers of CD1d<sup>hi</sup>CD5<sup>+</sup> B cells (A) or IL-10<sup>+</sup> B10 cells (B) from control (open bars) and CD22 mAb treated (closed bars) mice. (<b>C–D</b>) B10 cells regulate IgG Ab responses. WT mice were given CD22 or control mAb (<i>n</i> = 3 per group) on day 0, and immunized with DNP-KLH without adjuvant on days 0 and 21. (C) Serum DNP-specific Abs were quantified by ELISA. (D) The frequency of B cells secreting DNP-specific IgG was determined by ELISPOT analysis of spleen cells harvested on day 28. (A–D) Significant differences between control and CD22 mAb-treated mice (≥3 mice for each treatment group) are indicated: *p<0.05; **p<0.01.</p
Lymphocyte subsets in WT, CD22<sup>−/−</sup>, CD154<sup>TG</sup>, and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice<sup>a</sup>.
a<p>Numbers represent mean (±SEM) values from ≥4 littermates of each genotype. Asterisks indicate values significantly different from WT mice (*p<0.05; **p<0.01). PLN, peripheral LN; MLN, mesenteric LN; PC, peritoneal cavity.</p>b<p>B10 and B10pro cells were identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022464#pone-0022464-g003" target="_blank">Figure 3</a>.</p>c<p>IL-10-competent T cells were identified following 5 h stimulation with PMA/ionomycin.</p
CD154<sup>TG</sup>CD22<sup>−/−</sup> B10 cells are regulatory.
<p>(<b>A</b>) B10 cells in CD154<sup>TG</sup>CD22<sup>−/−</sup> mice are found predominately within the spleen CD1d<sup>hi</sup>CD5<sup>+</sup> B cell population. Splenocytes from WT and CD154<sup>TG</sup>CD22<sup>−/−</sup> mice were cultured for 5 h with L+PIM to induce IL-10 expression, with the cells analyzed for cell surface CD19, CD1d and CD5, and intracellular IL-10 expression. CD19<sup>+</sup>IL-10<sup>+</sup> and CD19<sup>+</sup>IL-10<sup>−</sup> B cells (left panels, gated regions) were further gated to show relative CD1d and CD5 expression (merged dot plots, right panels). Bar graphs show the mean (±SEM) B10 cell frequencies within the indicated populations for ≥4 mice of each genotype based on the gated regions indicated in the merged dot plots. Mean B10 cell frequencies significantly different between cell populations from the same genotype are indicated by asterisks (**p≤0.01), and for the same population between genotypes by crosses (††p<0.01). (<b>B</b>) Analysis of CD154<sup>TG</sup>CD22<sup>−/−</sup> spleen B cell purity within the CD1d<sup>hi</sup>CD5<sup>+</sup> (B10-rich) and CD1d<sup>lo</sup>CD5<sup>−</sup> populations following cell sorting of splenocytes stained for CD19, CD1d and CD5. These cells were subsequently used for the adoptive transfer experiments described in (C). (<b>C</b>) B10 cells from CD154<sup>TG</sup>CD22<sup>−/−</sup> mice reduce EAE disease severity. Purified CD1d<sup>hi</sup>CD5<sup>+</sup> or CD1d<sup>lo</sup>CD5<sup>−</sup> spleen B cells from naïve CD154<sup>TG</sup>CD22<sup>−/−</sup> mice (B) were either adoptively transferred into WT recipient mice immediately (non-activated) or were cultured with agonistic CD40 mAb for 48 h with LPS added during the final 5 h of culture before transfer. Other recipient mice received PBS alone (Control). One day after cell transfers, EAE was induced by MOG immunization. Values represent mean (±SEM) clinical EAE scores from 5 mice per group, with significant differences from PBS control mice indicated: *p<0.05.</p
Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin
<div><p>Background</p><p>Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy.</p><p>Methods and Findings</p><p>The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in <i>ex vivo</i> lesional psoriatic skin.</p><p>Conclusions</p><p>Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.</p></div
Skin Resident Immune Cell Activation (sRICA) leads to pro-inflammatory cytokine responses that are reduced by GSK2981278.
<p>(A) Skin explants were cultured ≥4 days under the indicated conditions. Explants were analyzed for tissue integrity by H&E. (B) Samples were pre-treated with 10 μM compound (closed bar) or vehicle (DMSO; open bars–set to 100%) for 1 day prior to 24–48 hrs of Th17 stimulation. Relative transcript levels were determined by qRT-PCR. (C) Samples were treated as in B, then analyzed daily for tissue integrity by H&E. Images are representative at least 3 independent experiments. (D) Samples were treated as in B. Graphs show the mean percent maximum stimulation of 3 independent experiments. Significant inhibition was determined by Student’s <i>t</i> test. (*p≤0.05; **p≤0.01; ***p≤0.001).</p
GSK2981278 attenuates inflammation in a mouse model of psoriasis.
<p>(A) Mice were treated topically with placebo or 1% GSK2981278 (1278) in ointment, and with imiquimod (IMQ) or petrolatum (vehicle). At study’s end (day +9 of IMQ treatment), back skin was imaged and stained (H&E). (B) Mean epidermal thickness is shown across 6–9 mice per treatment group. (C-E) Changes to local cytokine expression was determined following topical application of 1% or 0.1% compound in a simple ethanolic solution (60:40 ethanol:water). (C) Description of study groups for panels D-E. Skin cytokine levels on day +3 (D) or day +9 (E). Data reflect the mean ± SEM gene expression level across 6–9 mice per treatment group. Significant inhibition was determined by Student’s <i>t</i> test. (*p≤0.05;**p≤0.01).</p
Suppression of Th17-type cytokine production following topical application.
<p><i>Ex vivo</i> human skin was cultured in Franz Cell chambers for a total of 48 hours. GSK2981278 was applied to the dry surface of the skin at time zero followed 24 hrs later by activation of skin resident immune cells under Th17 polarizing conditions. The experimental schema is shown in panel A. Skin sections were harvested after 24 hrs of stimulation (48 hrs of compound treatment) and analyzed for relative gene expression of <i>il17a</i> (B) or <i>il17f</i> (C). Data are shown as the percent maximum expression of each cytokine as compared to Th17-stimulated samples treated topically with vehicle only.</p