Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators.

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1ß. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.

IκBζ is a key player in the antipsoriatic effects of secukinumab.

BACKGROUND: IκBζ plays a key role in psoriasis by mediating IL-17A-driven effects, but the molecular mechanism by which IL-17A regulates IκBζ expression is not clarified. OBJECTIVE: We sought to explore the molecular transformation in patients with psoriasis during anti-IL-17A (secukinumab) treatment with a focus on IκBζ. METHODS: The study was an open-label, single-arm, single-center secukinumab treatment study that included 14 patients with plaque psoriasis. Skin biopsy specimens and blood samples were collected on days 0, 4, 14, 42, and 84 and processed for microarray gene expression analysis. Furthermore, in vitro experiments with human keratinocytes and synovial fibroblasts were conducted. RESULTS: Secukinumab improved clinical scores and histologic psoriasis features. Moreover, secukinumab altered the skin transcriptome. The major transcriptional shift appeared between day 14 and day 42 after treatment initiation, although 80 genes were differentially expressed already at day 4. Expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IκB) ζ (NFKBIZ, the gene encoding IκBζ) was reduced already after 4 days of treatment in the skin. NFKBIZ expression correlated to Psoriasis Area and Severity Index score, and NFKBIZ mRNA levels in the skin decreased during anti-IL-17A treatment. Moreover, specific NFKBIZ signature genes were significantly altered during anti-IL-17A treatment. Finally, we identified NF-κB activator 1 (Act1), p38 mitogen-activated protein kinase (MAPK), Jun NH2-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as key signaling pathways in NFKBIZ/IκBζ regulation. CONCLUSION: Our results define a crucial role for IκBζ in the antipsoriatic effect of secukinumab. Because IκBζ signature genes were regulated already after 4 days of treatment, this strongly indicates that IκBζ plays a crucial role in the antipsoriatic effects mediated by anti-IL-17A treatment.

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1ß; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1ß pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1ß antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1ß in lung granuloma, and increased IL-1ß release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1ß antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1ß pathway activation in sarcoidosis and suggest both as valid therapeutic targets.

Application of clinical and molecular profiling data to improve patient outcomes in psoriatic arthritis.

Achieving a good outcome for a person with Psoriatic Arthritis (PsA) is made difficult by late diagnosis, heterogenous clinical disease expression and in many cases, failure to adequately suppress inflammatory disease features. Single-centre studies have certainly contributed to our understanding of disease pathogenesis, but to adequately address the major areas of unmet need, multi-partner, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient-representative organizations. In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for better short-term and long-term outcomes.

Improving outcomes in Psoriatic Arthritis Psoriatic Arthritis (PsA) is a form of arthritis which is found in approximately 30% of people who have the skin condition, Psoriasis. Frequently debilitating and progressive, achieving a good outcome for a person with PsA is made difficult by late diagnosis, disease clinical features and in many cases, failure to adequately control features of inflammation. Research studies from individual centres have certainly contributed to our understanding of why people develop PsA but to adequately address the major areas of unmet need, multi-centre, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient representative organisations (see appendix). In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. The participation of patient research partners in all stages of the work of HIPPOCRATES is highlighted. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for improvements in short-term and long-term outcomes.

Secukinumab for the treatment of psoriasis, psoriatic arthritis, and axial spondyloarthritis: Physical and pharmacological properties underlie the observed clinical efficacy and safety.

Psoriasis, psoriatic arthritis, and axial spondyloarthritis are systemic inflammatory diseases, each commonly manifesting as a spectrum of symptoms, complications, and comorbidities that arise differently in individual patients. Drugs targeting inflammatory cytokines common to the pathogenesis of each of these conditions have been developed, although their specific actions in the different tissues involved are variable. For a drug to be effective, it must be efficiently delivered to and locally bioactive in disease-relevant tissues. Detailed clinical data shed light on the therapeutic effects of individual biologics on specific domains or clinical manifestations of disease and assist in guiding treatment decisions. Pharmacologic, molecular, and functional properties of drugs strongly impact their observed safety and efficacy, and an understanding of these properties provides complementary insight. Secukinumab, a fully human monoclonal IgG1/κ antibody selectively targeting interleukin (IL)-17A, has been in clinical use for >6 years in the treatment of moderate to severe psoriasis, psoriatic arthritis, and both radiographic (also known as ankylosing spondylitis) and nonradiographic axial spondyloarthritis. In this review, we discuss pharmacokinetic and pharmacodynamic data for secukinumab to introduce clinicians to the pharmacological properties of this widely used drug. Understanding how these properties affect the observed clinical efficacy, safety, and tolerability of this drug in the treatment of IL-17A-mediated systemic inflammatory diseases is important for all physicians treating these conditions.

Nonhematopoietic IRAK1 drives arthritis via neutrophil chemoattractants.

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part I.

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

Novel 3-aminopyrazole inhibitors of MK-2 discovered by scaffold hopping strategy.

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II.

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D-E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC(50)<3 nM and inhibit the release of TNFalpha (IC(50)<0.3 microM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC(50)=0.05-0.23 microM), less potent in cells (IC(50)<1.1 microM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

Pyrrolo-pyrimidones: a novel class of MK2 inhibitors with potent cellular activity.

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.

Modulating ADME Properties by Fluorination: MK2 Inhibitors with Improved Oral Exposure.

MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.

IL-17A and Multiple Sclerosis: Signaling Pathways, Producing Cells and Target Cells in the Central Nervous System.

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). The importance of immune cells to MS pathology is supported by clinical data linking the depletion of T and B cells, or the prevention of their migration into the brain with significant reduction in relapses and development of new lesions. In vitro studies, preclinical animal models and encouraging data with the anti-IL-17A antibody secukinumab in a small proof of concept study in man, indicate that IL-17A, a key interleukin associated with many inflammatory and autoimmune diseases, may be involved in MS. Not only cells involved in adaptive immune responses such as Th17 cells and cytotoxic T cells, or innate immune responses such as mucosa-associated invariant T (MAIT) cells and γδT cells, but also CNS resident cells such as astrocytes and oligodendrocytes might contribute to the local production of IL-17A. IL-17A synergizes with other proinflammatory cytokines, by inducing the release of additional cytokines, mediators of tissue damage and chemokines, that recruit new inflammatory cells. IL-17A adversely affects the functions of microglia, astrocytes, oligodendrocytes, neurons, neural precursor cells and endothelial cells. Blockade of IL-17A might be beneficial to MS patients not only by inhibiting inflammation and tissue destruction, but also by enhancing repair processes.

Low-molecular-weight MK2 inhibitors: a tough nut to crack!

The p38 pathway has been at the center of interest for anti-inflammatory drug discovery for many years as it is crucial for the biosynthesis of TNF-α, IL-1ß and other mediators. Most of the anti-inflammatory effects of p38 inhibition are mediated through MAPK-activated protein kinase-2 (MK2), a direct downstream target of p38, which makes MK2 a very interesting drug target. Within the last 5 years, several classes of low-molecular-weight MK2 inhibitors were disclosed in the patent and primary literature. Advanced compounds could be optimized to nanomolar potencies and inhibit TNF-α release, as well as the phosphorylation of the MK2 substrate heat-shock protein 27 in cellular assays. This article will review the recent progress in this field and will highlight and discuss the most promising compound series disclosed so far.