Discovery of the Clinical Candidate IDOR-1117-2520: A Potent and Selective Antagonist of CCR6 for Autoimmune Diseases.

Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.

In Vitro Characterization of Sphingosine 1-Phosphate Receptor 1 (S1P1) Expression and Mediated Migration of Primary Human T and B Cells in the Context of Cenerimod, a Novel, Selective S1P1 Receptor Modulator.

Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P1) modulator currently investigated in a Phase IIb study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P1 receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P1 receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P1 re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P1 receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.

Mitogen-activated protein kinase signaling in childhood asthma development and environment-mediated protection.

BACKGROUND: While childhood asthma prevalence is rising in Westernized countries, farm children are protected. The mitogen-activated protein kinase (MAPK) pathway with its negative regulator dual-specificity phosphatase-1 (DUSP1) is presumably associated with asthma development. OBJECTIVES: We aimed to investigate the role of MAPK signaling in childhood asthma and its environment-mediated protection, including a representative selection of 232 out of 1062 children from two cross-sectional cohorts and one birth cohort study. METHODS: Peripheral blood mononuclear cells (PBMC) from asthmatic and healthy children were cultured upon stimulation with farm-dust extracts or lipopolysaccharide. In subgroups, gene expression was analyzed by qPCR (PBMCs, cord blood) and NanoString technology (dendritic cells). Protein expression of phosphorylated MAPKs was measured by mass cytometry. Histone acetylation was investigated by chromatin immunoprecipitation. RESULTS: Asthmatic children expressed significantly less DUSP1 (p = .006) with reduced acetylation at histone H4 (p = .012) compared with healthy controls. Farm-dust stimulation upregulated DUSP1 expression reaching healthy levels and downregulated inflammatory MAPKs on gene and protein levels (PBMCs; p ≤ .01). Single-cell protein analysis revealed downregulated pMAPKs upon farm-dust stimulation in B cells, NK cells, monocytes, and T-cell subpopulations. CONCLUSION: Lower DUSP1 baseline levels in asthmatic children and anti-inflammatory regulation of MAPK in several immune cell types by farm-dust stimulation indicate a regulatory function for DUSP1 for future therapy contributing to anti-inflammatory characteristics of farming environments.

Restoration of Natural Killer Cell Antimetastatic Activity by IL12 and Checkpoint Blockade.

Immune checkpoint therapies target tumor antigen-specific T cells, but less is known about their effects on natural killer (NK) cells, which help control metastasis. In studying the development of lung metastases, we found that NK cells lose their cytotoxic capacity and acquire a molecular signature defined by the expression of coinhibitory receptors. In an effort to overcome this suppressive mechanism, we evaluated NK cell responses to the immunostimulatory cytokine IL12. Exposure to IL12 rescued the cytotoxicity of NK cells but also led to the emergence of an immature NK cell population that expressed high levels of the coinhibitory molecules PD-1, Lag-3, and TIGIT, thereby limiting NK cell-mediated control of pulmonary metastases. Notably, checkpoint blockade therapy synergized with IL12 to fully enable tumor control by NK cells, demonstrating that checkpoint blockers are not only applicable to enhance T cell-mediated immunotherapy, but also to restore the tumor-suppressive capacity of NK cells. Cancer Res; 77(24); 7059-71. ©2017 AACR.

IL-12 protects from psoriasiform skin inflammation.

Neutralization of the common p40-subunit of IL-12/23 in psoriasis patients has led to a breakthrough in the management of moderate to severe disease. Aside from neutralizing IL-23, which is thought to be responsible for the curative effect, anti-p40 therapy also interferes with IL-12 signalling and type 1 immunity. Here we dissect the individual contribution of these two cytokines to the formation of psoriatic lesions and understand the effect of therapeutic co-targeting of IL-12 and IL-23 in psoriasis. Using a preclinical model for psoriatic plaque formation we show that IL-12, in contrast to IL-23, has a regulatory function by restraining the invasion of an IL-17-committed γδT (γδT17) cell subset. We discover that IL-12 receptor signalling in keratinocytes initiates a protective transcriptional programme that limits skin inflammation, suggesting that collateral targeting of IL-12 by anti-p40 monoclonal antibodies is counterproductive in the therapy of psoriasis.

IL17A-Mediated Endothelial Breach Promotes Metastasis Formation.

The role of the IL23/IL17A axis in tumor-immune interactions is a matter of controversy. Although some suggest that IL17A-producing T cells (TH17) can suppress tumor growth, others report that IL17A and IL23 accelerate tumor growth. Here, we systematically assessed the impact of IL17A-secreting lymphocytes in several murine models of tumor lung metastasis. Genetic fate mapping revealed that IL17A was secreted within lung metastases predominantly by γδ T cells, whereas TH17 cells were virtually absent. Using different tumor models, we found Il17a(-/-) mice to consistently develop fewer pulmonary tumor colonies. IL17A specifically increased blood vessel permeability and the expression of E-selectin and VCAM-1 by lung endothelial cells in vivo. In transgenic mice, specific targeting of IL17A to the endothelium increased the number of tumor foci. Moreover, the direct impact of IL17A on lung endothelial cells resulted in impaired endothelial barrier integrity, showing that IL17A promotes the formation of lung metastases through tumor-endothelial transmigration.

Dermal IL-17-producing γδ T cells establish long-lived memory in the skin.

Conventional αß T cells have the ability to form a long-lasting resident memory T-cell (TRM ) population in nonlymphoid tissues after encountering foreign antigen. Conversely, the concept of 'innate memory', where the ability of nonadaptive branches of the immune system to deliver a rapid, strengthened immune response upon reinfection or rechallenge, is just emerging. Using the αß T-cell-independent Aldara psoriasis mouse model in combination with genetic fate-mapping and reporter systems, we identified a subset of γδ T cells in mice that is capable of establishing a long-lived memory population in the skin. IL-17A/F-producing Vγ4(+) Vδ4(+) T cells populate and persist in the dermis for long periods of time after initial stimulation with Aldara. Experienced Vγ4(+) Vδ4(+) cells show enhanced effector functions and mediate an exacerbated secondary inflammatory response. In addition to identifying a unique feature of γδ T cells during inflammation, our results have direct relevance to the human disease as this quasi-innate memory provides a mechanistic insight into relapses and chronification of psoriasis.

Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

IL-12-and IL-23 in health and disease.

Interleukin (IL)-12 and IL-23 play important roles in the development of experimental autoimmune disease models and numerous afflictions affecting humans. Preclinical data over the last 20 years combined with successful clinical trials has identified a clear relationship between IL-12, IL-23 and the generation of pathogenic T helper cells capable of orchestrating tissue inflammation. Observations made in the clinic have shown that IL-12p40, a common subunit shared by IL-12 and IL-23, is critical to pathologies associated with psoriasis, inflammatory bowel disease (IBD) and tumor growth. These advancements have set in motion the development of a number of potential therapeutics aimed at manipulating IL-12/23 signaling pathways in both mice and humans. This review will discuss a brief history of the understanding and expansion of the IL-12 cytokine family, some difficulties associated with preclinical data interpretation and finally the medicinal interventions that have been developed to combat IL-12/23-driven autoimmune disorders.

The role of metalloproteinase ADAM17 in regulating ICOS ligand-mediated humoral immune responses.

Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17(ex/ex)) mice, in which ADAM17 expression is reduced by 90-95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17(ex/ex) mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.

Intratumoral IL-12 combined with CTLA-4 blockade elicits T cell-mediated glioma rejection.

Glioblastomas (GBs) are the most aggressive form of primary brain cancer and virtually incurable. Accumulation of regulatory T (T reg) cells in GBs is thought to contribute to the dampening of antitumor immunity. Using a syngeneic mouse model for GB, we tested whether local delivery of cytokines could render the immunosuppressive GB microenvironment conducive to an antitumor immune response. IL-12 but not IL-23 reversed GB-induced immunosuppression and led to tumor clearance. In contrast to models of skin or lung cancer, IL-12-mediated glioma rejection was T cell dependent and elicited potent immunological memory. To translate these findings into a clinically relevant setting, we allowed for GB progression before initiating therapy. Combined intratumoral IL-12 application with systemic blockade of the co-inhibitory receptor CTLA-4 on T cells led to tumor eradication even at advanced disease stages where monotherapy with either IL-12 or CTLA-4 blockade failed. The combination of IL-12 and CTLA-4 blockade acts predominantly on CD4(+) cells, causing a drastic decrease in FoxP3(+) T reg cells and an increase in effector T (T eff) cells. Our data provide compelling preclinical findings warranting swift translation into clinical trials in GB and represent a promising approach to increase response rates of CTLA-4 blockade in solid tumors.

Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice.

Psoriasis is a common, relapsing inflammatory skin disease characterized by erythematous scaly plaques. Histological manifestations of psoriasis include keratinocyte dysregulation and hyperproliferation, elongated rete ridges, and inflammatory infiltrates consisting of T cells, macrophages, dendritic cells, and neutrophils. Despite the availability of new effective drugs to treat psoriasis, the underlying mechanisms of pathogenesis are still poorly understood. Recent studies have shown that Aldara cream, used to treat benign skin abnormalities, triggers psoriasis-like disease in humans and mice and have implicated Th17 cells in disease initiation. Using this as a model, we found a predominant role for the Th17 signature cytokines IL-17A, IL-17F, and IL-22 in psoriasiform plaque formation in mice. Using gene-targeted mice, we observed that loss of Il17a, Il17f, or Il22 strongly reduced disease the severity of psoriasis. However, we found that Th17 cells were not the primary source of these pathogenic cytokines. Rather, IL-17A, IL-17F, and IL-22 were produced by a skin-invading population of γδ T cells and RORγt(+) innate lymphocytes. Furthermore, our findings establish that RORγt(+) innate lymphocytes and γδ T cells are necessary and sufficient for psoriatic plaque formation in an experimental disease model that closely resembles human psoriatic plaque formation.

Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins.

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

Recently T-helper 17 (Th17) cells were demonstrated to disrupt the blood-brain barrier (BBB) by the action of IL-17A. The aim of the present study was to examine the mechanisms that underlie IL-17A-induced BBB breakdown. Barrier integrity was analyzed in the murine brain endothelial cell line bEnd.3 by measuring the electrical resistance values using electrical call impedance sensing technology. Furthermore, in-cell Western blots, fluorescence imaging, and monocyte adhesion and transendothelial migration assays were performed. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. IL-17A induced NADPH oxidase- or xanthine oxidase-dependent reactive oxygen species (ROS) production. The resulting oxidative stress activated the endothelial contractile machinery, which was accompanied by a down-regulation of the tight junction molecule occludin. Blocking either ROS formation or myosin light chain phosphorylation or applying IL-17A-neutralizing antibodies prevented IL-17A-induced BBB disruption. Treatment of mice with EAE using ML-7, an inhibitor of the myosin light chain kinase, resulted in less BBB disruption at the spinal cord and less infiltration of lymphocytes via the BBB and subsequently reduced the clinical characteristics of EAE. These observations indicate that IL-17A accounts for a crucial step in the development of EAE by impairing the integrity of the BBB, involving augmented production of ROS.-Huppert, J., Closhen, D., Croxford, A., White, R., Kulig, P., Pietrowski, E., Bechmann, I., Becher, B., Luhmann, H. J., Waisman, A., Kuhlmann, C. R. W. Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

[Plasmacytoid dendritic cells in pathophysiology of immune responses]. / Plazmocytoidalne komórki dendrytyczne w patofizjologii procesów odpornosciowych.

Plasmacytoid dendritic cells (pDC), so named because of histological similarities to antibody-producing plasma cells, comprise a unique dendritic cell population. Despite their low numbers in blood they play a key role in anti-viral immunity, mainly through secreting massive amounts of type 1 interferon following an engagement of their Toll-like receptors, such as TLR7 and TLR9, by viral nucleic acids. In a mature state, pDC display a propensity to present antigens to naive lymphocytes. Through their unique potential to rapidly and efficiently secrete type I interferon and serve as antigen presenting cells, pDC are currently considered as one of the major regulators of immune responses.

Staphylococcus aureus-derived staphopain B, a potent cysteine protease activator of plasma chemerin.

Chemerin is an attractant for cells that express the serpentine receptor CMKLR1, which include immature plasmacytoid dendritic cells (pDC) and macrophages. Chemerin circulates in the blood where it exhibits low biological activity, but upon proteolytic cleavage of its C terminus, it is converted to a potent chemoattractant. Enzymes that contribute to this conversion include host serine proteases of the coagulation, fibrinolytic, and inflammatory cascades, and it has been postulated that recruitment of pDC and macrophages by chemerin may serve to balance local tissue immune and inflammatory responses. In this work, we describe a potent, pathogen-derived proteolytic activity capable of chemerin activation. This activity is mediated by staphopain B (SspB), a cysteine protease secreted by Staphylococcus aureus. Chemerin activation is triggered by growth medium of clinical isolates of SspB-positive S. aureus, but not by that of a SspB(null) mutant. C-terminal processing by SspB generates a chemerin isoform identical with the active endogenous attractant isolated from human ascites fluid. Interestingly, SspB is a potent trigger of chemerin even in the presence of plasma inhibitors. SspB may help direct the recruitment of specialized host cells, including immunoregulatory pDC and/or macrophages, contributing to the ability of S. aureus to elicit and maintain a chronic inflammatory state.

Regulation of the release and function of tumor cell-derived soluble CD44.

CD44, a major receptor for glycosaminoglycan hyaluronan (HA), is a broadly distributed cell surface glycoprotein implicated in multiple functions, including tumor growth and dissemination. The affinity of surface CD44 for HA is subject to regulation at several levels. CD44 is found in multiple phases, including as an integral transmembrane protein and as soluble fragment of the extracellular domain found in the circulation and other body fluids. Transmembrane CD44 and its ability to interact with HA have been a focus of numerous studies in the past, but the function of soluble CD44 remains obscure. Interestingly, malignant diseases are often associated with an increase in the plasma level of CD44. The delineation of the HA binding capacity of tumor-derived soluble CD44 is an important step toward understanding the biological function of this molecule. In this study, we demonstrate that tumor cells activated to bind HA by cytokines rapidly release CD44 upon treatment with phorbol ester (PMA). The affinity for HA of the soluble CD44 released in response to PMA varied depending on the cytokine pretreatment. These results suggest that the function of tumor-derived soluble CD44, like the transmembrane form of the receptor, can be regulated.

Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades.

Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the "rapid responder" leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation.

Acute phase mediator oncostatin M regulates affinity of alpha1-protease inhibitor for concanavalin A in hepatoma-derived but not lung-derived epithelial cells.

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.

Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.

XR5118 [(3 Z,6 Z )-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P(1) residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central beta-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.