14 research outputs found
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
Treatment of psoriatic tissue with the RORγ inverse agonist GSK2981278 reduces proinflammatory cytokine levels.
<p>Three psoriatic skin biopsies were obtained via 3-4mm punch biopsy and placed in Unisol buffer for overnight shipment. Upon arrival, biopsy sections were placed in Cornification media without stimulation for 12–14 hours with either 0.2% DMSO or 10 μM compound. The percent inhibition of each biomarker compared to culture with DMSO alone is shown. Each point represents an individual patient sample. The percent maximum expression ± SEM for each analyte assayed of each tissue was calculated relative to time zero. Significant inhibition was determined by Paired <i>t</i>-test. (**p≤0.01).</p
MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses
<div><p>CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4<sup>+</sup> and CD8<sup>+</sup> T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. <i>In vivo</i>, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses <i>in vivo</i>.</p></div
MALT1 protease-dead mice develop inflammatory cell infiltrates in multiple organs.
<p>H&E-stained histological sections of tissues from MALT1 <i>Wt</i> (left panels A, C, E, G; n = 3) and <i>Malt1</i><sup>PD/PD</sup> (right panels B, D, F H; n = 6) mice. Results from <i>Malt1</i><sup>-/-</sup> (n = 3) and <i>Malt1</i><sup>+/PD</sup> (n = 2) mice were identical to <i>Wt</i> mice. (A, B) Lung at 100X magnification. Airways of <i>Wt</i> and <i>Malt1</i><sup>PD/PD</sup> mice, including the B, bronchiolar (B) and terminal airways (TA) were relatively clear of inflammatory infiltrates. Distinctive findings were present in the lungs of <i>Malt1</i><sup>PD/PD</sup> mice, which featured pulmonary veins (V) surrounded by a prominent mixed mononuclear inflammatory cell infiltrate predominated by lymphocytes and histiocytic cells (inset, 400X magnification). (C, D) Limbic region of eye (150X magnification). Cornea from <i>Wt</i> eye demonstrates typical anatomical features of a thin corneal epithelium (CE) overlying the substantia propria (SP). Occasional linear nuclei in the SP are part of the normal fibroblast populations. Sclera (S), pigmented iris (I) and lens (L) are normal in <i>Malt1</i><sup>PD/PD</sup> mice. Cornea from <i>Malt1</i><sup>PD/PD</sup> mice consistently had neutrophilic inflammatory cell infiltrates (arrows) of varying severity in the substantia propria of the cornea. This particular <i>Malt1</i><sup>PD/PD</sup> mouse also had a florid neutrophilic infiltrate towards the central portion of the cornea (inset, 150X magnification). (E, F) Non-glandular stomach (50X magnification). Normal non-glandular stomach was comprised of stratified squamous epithelium (Ep) with a small amount of keratinized stratum corneum (Sc) and only occasional inflammatory cells evident in the submucosa (Sub). Non-glandular stomach from <i>Malt1</i><sup>PD/PD</sup> mice demonstrated marked hyperkeratosis of the stratum corneum and prominent hyperplasia of the epithelium. A mixed inflammatory infiltrate was commonly present in the mural wall, with this particular mouse having a moderate neutrophilic infiltrate in the submucosa (arrow). (G, H) Glandular stomach (50X magnification). The fundic mucosa (Muc) of the <i>Wt</i> mouse is regular, even, and is formed by undulating gastric pits. The relatively acellular tunica adventitia (Ta) is the thin tissue layer (arrow) sandwiched between the thicker tunica muscularis (Tm) and the adipose tissue (Ad) / pancreas (Pan). Reactive hyperplasia of the glandular mucosa was present in <i>Malt1</i><sup>PD/PD</sup> mice, and characterized by prominent thickening of the mucosa and loss of regular and even mucosal architecture. Mixed inflammatory cell infiltrates were increased in the mural wall of stomachs from <i>Malt1</i><sup>PD/PD</sup> mice, with this particular mouse demonstrating an intense inflammatory infiltrate of the tunica adventicia that is predominated by populations of lymphocytes and histiocytic cells.</p
The development of MZ and B1 B cells populations is dependent upon MALT1 protease activity.
<p>Representative flow cytometry analysis of lymphocyte populations in MALT1 <i>Wt</i>, <i>Malt1</i><sup>-/-</sup>, and <i>Malt1</i><sup>PD/PD</sup> mice. (A) CD4<sup>+</sup>CD8a<sup>+</sup>, CD4<sup>+</sup>CD8a<sup>-</sup>, and CD4<sup>-</sup>CD8a<sup>+</sup> T cell populations in the thymus. (B) CD4<sup>+</sup>CD8a<sup>+</sup>, CD4<sup>+</sup>CD8a<sup>-</sup>, and CD4<sup>-</sup>CD8a<sup>+</sup> T cell populations in the spleen. (C) Mature follicular (IgD<sup>hi</sup>IgM<sup>lo</sup>), transitional T1 (IgD<sup>lo</sup>IgM<sup>hi</sup>), and T2 (IgD<sup>hi</sup>IgM<sup>hi</sup>) B cell populations in the spleen. (D, F) MZ B cell (CD21<sup>-</sup>CD23<sup>+</sup>) population in the spleen. (E, G) B1 B cell (IgM<sup>hi</sup>CD5<sup>lo</sup>) population in peritoneal fluid. Bar graphs represent average cell numbers for 3–4 mice/group. Significance was determined relative to the <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01. Error bars represent +/- SEM.</p
MALT1 protease activity is required for TCR- but not BCR-mediated lymphocyte activation.
<p>(A) CD4<sup>+</sup> T cells and (B) CD8<sup>+</sup> T cells from the spleens of <i>Wt</i>, <i>Malt1</i><sup>-/-</sup>, and <i>Malt1</i><sup>PD/PD</sup> mice were activated by co-stimulation with anti-CD3 and anti-CD28 beads, and proliferation was measured after 96 h by <sup>3</sup>H-thymidine incorporation, and IL-2 production after 24 h by MSD. The total IL-2 produced by CD4<sup>+</sup> and CD8<sup>+</sup> T cells from <i>Wt</i> mice was approximately 2–3 ng/ml and 100–400 pg/ml, respectively. Where indicated, T cells were treated with 126 μM of the MALT1 inhibitor Z-VRPR-FMK (VRPR) for 45 min prior to stimulation. (C) B220<sup>+</sup> B cells from the spleens of <i>Wt</i>, <i>Malt1</i><sup>-/-</sup>, and <i>Malt1</i><sup>PD/PD</sup> mice were stimulated with 20 μg/ml anti-IgM or 20 μg/ml LPS and proliferation was measured after 96 h by <sup>3</sup>H-thymidine incorporation. Bar graphs from Fig 3A-C represent the average response expressed as a percent of the stimulated <i>Wt</i> control (LPS treatment for B220<sup>+</sup> cells) for 4–6 mice/group, and are representative of 3 or more studies. (D) Splenic B220<sup>+</sup> B cells from <i>Wt</i> and <i>Malt1</i><sup>PD/PD</sup> mice were labeled with CFSE prior to stimulation with anti-IgM and proliferation was analyzed by FACS three days post stimulation. <i>Wt</i> EC50 = 2.6 μg/ml; <i>Malt1</i><sup>PD/PD</sup> EC50 = 6.4 μg/ml. Each data point represents the average response of five mice expressed as a percent of <i>Wt</i> proliferation at the highest dose. (E) Splenic B220+ B cells were either unstimulated or (F) stimulated with α-IgM (20 μg/ml) for three days and viability was analyzed by FACS. Each bar is an average from five mice. Significance was determined relative to the stimulated <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001, ‡<i>p</i> < 0.0001. Error bars represent +/- SEM.</p
Mixed inflammatory infiltrates of T cells, B cells, and macrophages in MALT1 protease-dead mice.
<p>IHC photomicrographs (80X magnification, insets 400X magnification) of inflammatory cell infiltrates surrounding vessels (V) of the lung and tunica adventicia of glandular stomach in <i>Malt1</i><sup>PD/PD</sup> mice, using antibodies against: (A, D) CD3 to detect T cells; (B, E) CD45 to detect B-cell; and (C, F) F4/80 to detect macrophage. Non-selective background staining was higher with the F4/80 antibody, as evident with the pale staining of erythrocytes within the vessel lumen (V, Fig 8C). Therefore, macrophage identification was made by considering the higher staining intensity and morphology of cells stained (inset, Fig 8C). IHC photomicrographs of inflammatory cell infiltrates in lung of <i>Malt1</i><sup>PD/PD</sup> mice (8A, C) demonstrates a mixed population of T-cells, B-cells and macrophages comprising the perivascular cuffs of pulmonary vessels. Similar to the lung infiltrates of <i>Malt1</i><sup>PD/PD</sup>, inflammatory infiltrates in deeper glandular portions of stomach had mixed population of T cells, B cells and macrophages in the tunica adventicia (Fig 8D, F).</p