IL-25 regulates Th17 function in autoimmune inflammation

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17–producing T cells. We have generated IL-25–deficient (il25−/−) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25−/− mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17–, IFNγ-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNγ in il25−/− mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1β, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity

Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation

The C-type lectin-like receptor CD161, which has recently been described to promote T cell expansion, is expressed on a discrete subset of human CD4 T cells. The function of such cells, however, has remained elusive. We now demonstrate that CD161+ CD4 T cells comprise a circulating and gut-resident T helper 17 (Th17) cell population. During Crohn's disease (CD), these CD161+ cells display an activated Th17 phenotype, as indicated by increased expression of interleukin (IL)-17, IL-22, and IL-23 receptor. CD161+ CD4 T cells from CD patients readily produce IL-17 and interferon γ upon stimulation with IL-23, whereas, in healthy subjects, priming by additional inflammatory stimuli such as IL-1β was required to enable IL-23–induced cytokine release. Circulating CD161+ Th17 cells are imprinted for gut homing, as indicated by high levels of CC chemokine receptor 6 and integrin β7 expression. Supporting their colitogenic phenotype, CD161+ Th17 cells were found in increased numbers in the inflammatory infiltrate of CD lesions and induced expression of inflammatory mediators by intestinal cells. Our data identify CD161+ CD4 T cells as a resting Th17 pool that can be activated by IL-23 and mediate destructive tissue inflammation

Development of Gut-Selective Pan-Janus Kinase Inhibitor TD-1473 for Ulcerative Colitis: A Translational Medicine Programme.

Background and aimsOral systemic pan-Janus kinase [JAK] inhibition is effective for ulcerative colitis [UC] but is limited by toxicities. We describe preclinical to clinical translation of TD-1473-an oral gut-selective pan-JAK inhibitor-from in vitro characterization through a Phase 1b study in patients with UC.MethodsTD-1473 JAK inhibition potency was evaluated in vitro; plasma pharmacokinetics, safety and efficacy were assessed in mice. In a first-time-in-human study, plasma pharmacokinetics and safety were assessed after single and multiple [14 days] ascending doses administered orally to healthy subjects. The Phase 1b study randomized patients with moderately to severely active UC to receive once-daily oral TD-1473 20, 80 or 270 mg, or placebo for 28 days. Plasma and colonic tissue concentrations were measured; safety was assessed; and efficacy was evaluated by UC clinical parameters, disease-surrogate biomarkers, endoscopy, histology and colonic tissue JAK signalling.ResultsTD-1473 exhibited potent pan-JAK inhibitory activity in vitro. Oral TD-1473 administration to mice achieved high, biologically active colonic tissue concentrations with low plasma exposure and decreased oxazolone-induced colitis activity without reducing blood cell counts vs placebo. TD-1473 administration in healthy human subjects and patients with UC yielded low plasma exposure and was generally well tolerated; treatment in patients with UC resulted in biologically active colonic tissue concentrations and descriptive trends toward reduced clinical, endoscopic and histological disease activity vs placebo.ConclusionGut-selective pan-JAK inhibition with TD-1473 administration resulted in high intestinal vs plasma drug exposure, local target engagement, and trends toward reduced UC disease activity. [Clinicaltrials.gov NCT02657122, NCT02818686]

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system

<div><p>While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the ‘immune fingerprint’ of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.</p></div

IL-23 is essential for T cell–mediated colitis and promotes inflammation via IL-17 and IL-6

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10–deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases

The impact of small molecule inhibitors on human T cell function.

<p>The production of IL-2 as well as the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in a human <i>in vitro</i> PBMC assay. The production of IL-2 was evaluated 24h following stimulation with α-CD3 and α-CD28 while the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in the same cultures 72h following α-CD3 and α-CD28 stimulation. Small molecule inhibitors suppress α-CD3 and α-CD28 induced IL-2 production (A), CD4⁺ T cell proliferation (B) and CD8⁺ T cell proliferation (C) in human PBMCs. For each compound, the potency (red circles) is plotted along the top X-axis and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the bottom X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ ovalues. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound.</p

Impact of SYK/ZAP-70 inhibitor and prednisolone on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 3, 6 and 24 h following treatment with three different concentrations of SYK/ZAP-70 inhibitor and prednisolone. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. Transcript expression of IL-2 under unstimulated, stimulated, and stimulated plus SYK/ZAP-70 inhibitor or prednisolone treated conditions at the 3h (B), 6h (C) and 24h (D) time points in the T cell stimulation assay. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-D) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

The effect of small molecule inhibitors on reactive oxygen species (ROS) production by human phagocytes.

<p>Ten minutes following stimulation with opsonized bacteria, the production of ROS in neutrophils and monocytes from human whole blood was evaluated using flow cytometry. Small molecule inhibitors inhibit ROS production neutrophils (A) and monocytes (B) in human whole blood following stimulation with opsonized bacteria. For each compound, the potency (red circles) and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound. The positive control used in this assay (Diphenyliodonium chloride [DPI], an NADPH oxidase inhibitor), can inhibit ROS production from neutrophils (C) or monocytes (D) in a dose dependent manner. A BTK inhibitor consistently inhibited ~50% neutrophil ROS production (E) across multiple donors evaluated in this assay. Each point in the dose response curve indicates mean±SEM of % inhibition at that dose from n = 6–8 donors.</p

Immune function impact profiles of small molecule inhibitors.

<p>Small molecule inhibitors were evaluated in six different functional assays with nine different read-outs. For a given compound on the Y-axis, each symbol on the X-axis represents the protein binding corrected cellular potency of that compound in the corresponding assay. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values in the X-axis. The reported IC₅₀ values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound in each assay. Immune function impact of a broader set (A) or a subset (B) of inhibitors are presented.</p