Khusru Asadullah

Selective glucocorticoid receptor agonists (SEGRAs): novel ligands with an improved therapeutic index.

Glucocorticoids are among the most successful therapies in the treatment of chronic inflammatory and autoimmune diseases. Their efficacy seems to be caused by the interference of the ligand-activated glucocorticoid receptor with many pro-inflammatory pathways via different mechanisms. The ubiquitous expression of the glucocorticoid receptor is a prerequisite for efficacy. Their main drawback, however, is due to their potential to induce adverse effects, in particular upon high dosage and prolonged usage. For the purpose reducing systemic side effects, topical glucocorticoids that act locally have been developed. Nevertheless, undesirable cutaneous effects such as skin atrophy persist from the use of topical glucocorticoids. Therefore a high medical need exists for drugs as effective as glucocorticoids but with a reduced side effect profile. Glucocorticoids function by binding to and activating the glucocorticoid receptor which positively or negatively regulates the expression of specific genes. Several experiments suggest that negative regulation of gene expression by the glucocorticoid receptor accounts for its anti-inflammatory action. This occurs through direct or indirect binding of the receptor to pro-inflammatory transcription factors that are already bound to their regulatory sites. The positive action of the receptor occurs through homodimer binding of the ligand receptor complex to discrete nucleotide sequences and this contributes to some of the adverse effects of the hormone. Glucocorticoid receptor ligands that promote the negative regulatory action of the receptor with reduced positive regulatory function should therefore show an improved therapeutic index. A complete separation of the positive from the negative regulatory activities of the receptor has so far not been possible because of the interdependent nature of the two regulatory processes. Nevertheless, recent understanding of the molecular mechanisms of the GR has triggered several drug discovery programs and these have led to the identification of dissociated GR-ligands. Such selective GR agonists (SEGRAs) are likely to enter clinical testing soon.

The Th17 cytokine IL‐22 induces IL‐20 production in keratinocytes: A novel immunological cascade with potential relevance in psoriasis

Psoriasis is a common chronic skin disease. Recent studies demonstrated that IL‐20 and IL‐22, cytokines produced by keratinocytes and T cells, respectively, both inhibit keratinocyte terminal differentiation and induce psoriasis‐like epidermis alterations. Here, we investigated the relationship between these mediators. Although IL‐20 was not able to regulate IL‐22 production, IL‐22 induced IL‐20 mRNA and protein in human keratinocytes. However, IL‐22 had only a minimal effect, if any, on IL‐19 and IL‐26. Cutaneous IL‐20 was also elevated in mice following IL‐22 application. Accordingly, some of IL‐22s effects on differentiation‐regulating genes were partially mediated by an endogenous, secreted protein and attenuated by anti‐IL‐20 Ab. Like IL‐22, IL‐17A and TNF‐α induced IL‐20 in keratinocytes, whereas IFN‐γ and IL‐20 itself did not. Furthermore, IL‐17A and TNF‐α individually strengthened the IL‐22‐induced IL‐20 production. In lesional skin of psoriasis patients, highly elevated IL‐20 levels strongly correlated with IL‐22, and to a lesser extent, with IL‐17A and TNF‐α. As previously shown for IL‐22, IL‐20 blood levels correlated with the disease severity, although with a lower significance. This study demonstrates that a T‐cell mediator induces a tissue cell mediator with similar effects to its own and therefore suggests the existence of a novel type of pathogenetic cascade.

Heme oxygenase-1 inhibits T cell-dependent skin inflammation and differentiation and function of antigen-presenting cells

Abstract: Hemeu2003oxygenase‐1 (HO‐1) is increased in psoriatic skin. We asked for the impact of physiological and pharmacological HO‐1 induction on skin immunity and the mechanisms involved in HO‐1‐induced immunomodulation. We found cutaneous HO‐1 expression upregulated comparable with suppressors of cytokine signalling (SOCS)1 and SOCS3 in psoriasis and atopic eczema and temporarily increased in murine ovalbumin‐induced late phase reaction (LPR) and 2,4‐dinitrofluorobenzene (DNFB)‐induced contact hypersensitivity (CHS). Cutaneous inflammation was enhanced by HO‐1 inhibition and was abrogated by treatment with the HO‐1 inducer cobaltic protoporphyrin (CoPP) both when applied around sensitization or before challenge. HO‐1 inhibition specifically prevented the anti‐inflammatory CoPP effect. CoPP inhibited T cell proliferation in splenocytes of treated mice and in human mixed leukocyte reaction and lymphocyte transformation test. CoPP induced HO‐1 in antigen‐presenting cells and depressed monocytic accessory molecule expression and the differentiation and maturation of monocyte‐derived dendritic cells (MDDC). It decreased tumor necrosis factor (TNF)‐α and interleukin (IL)‐12 production while increasing IL‐10 secretion. The antigen‐presenting capacity was diminished in CoPP‐treated and HO‐1‐transduced MDDC. We demonstrate for the first time the physiological role of HO‐1 in the limitation of skin inflammation and implement pharmacological HO‐1 induction as a therapeutic approach for Tu2003cell‐dependent inflammatory dermatoses. Suppression of antigen‐presenting cells may represent a main anti‐inflammatory mechanism of HO‐1.

The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in the p38/TNF-α Pathway of Systemic and Cutaneous Inflammation

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-alpha, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-alpha levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-alpha and IL-1beta levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-alpha neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-alpha production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-alpha, has a rather elusive role in T-cell-dependent cutaneous inflammation.

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases

Abstract:u2002 T‐cell‐mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis, allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL‐2‐inducible tyrosine kinase (Itk), an enzyme acting downstream of the T‐cell receptor (TCR), in T‐cell‐dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor, Compound 44, created by chemical optimization of an initial high‐throughput screening hit, inhibited Itk’s activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition, our data reveal that human Itk, comparable to its murine homologue, is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally, silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR–induced lymphokine secretion. In conclusion, Itk represents an interesting new target for the therapy of T‐cell‐mediated inflammatory skin diseases.

CCR4 and CCR10 ligands play additive roles in mouse contact hypersensitivity

Abstract:u2002 Psoriasis, atopic dermatitis and allergic contact dermatitis are T‐cell‐mediated inflammatory skin diseases; chemokine receptors (CCR) 4 and 10 play an important role in the ligand‐mediated recruitment of T cells into the skin in mice and humans, specifically with regards to tethering, firm adhesion and subsequent extravasation to the sight of injury. We utilized established murine models of dinitrofluorobenzene‐, trimellitic acid anhydride‐ or oxazalone‐induced contact hypersensitivity, to reflect the various Th‐polarizations of different skin diseases, and investigated the functional effect of antibody blocking of single CCR ligands or combination therapy to block all CCR4 and CCR10 ligands. Our results indicate a greater reduction in inflammatory response – measured by oedema formation, myeloid cell and neutrophil infiltration and activity and CD3+ cell infiltration at the site of injury – with combination antibody therapy to CCR4 and CCR10 ligands versus controls, in nearly every tested condition. We conclude that blocking CCR4 and CCR10 simultaneously, or their ligands, should be beneficial in the treatment of T‐cell‐mediated skin diseases.

Immunomodulation by a novel, dissociated Vitamin D3 analogue

Abstract:u2002 The biologically active metabolite of vitamin D3, 1α,25‐dihydroxyvitamin D3, has potent immunomodulatory activity; however, its clinical use is limited because of its hypercalcaemic activity in anti‐inflammatory active doses. Here, we present ZK203278, a novel, structurally different vitamin D3 analogue with profound immunomodulatory activities. It potently inhibits lymphocyte proliferation in the mixed lymphocyte reaction, and release of cytokines that are central in inflammation, such as TNFα and IL‐12 in the low nanomolar range. Similarly, expression of cell‐surface molecules involved in cell adhesion and antigen presentation, e.g. to T cells, is down‐regulated on human monocytes by low nanomolar concentrations of ZK203278. Potent anti‐inflammatory activity has been demonstrated also in vivo in rodent disease models. ZK203278 inhibited allergic contact dermatitis in rodents after oral administration in doses approximately two orders of magnitude below the hypercalcaemic threshold dose. Moreover, ZK203278 significantly prolonged skin allograft survival in rats in well‐tolerated doses. Altogether ZK203278, in contrast to 1α,25‐dihydroxyvitamin D3, exerts considerable immunomodulatory activity at non‐hypercalcaemic dosages and may have therapeutic potential for immune disorders or transplant rejection.

Correction: RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

T cells play a central role in many inflammatory diseases, hence the identification and validation of T cell-specific target genes will increase the understanding of T cell function in pathologic inflammatory situations. RNA interference (RNAi), with its ability to induce specific gene silencing in mammalian cells, represents a powerful technology to investigate and validate the function of pharmaceutical target genes in vitro and in vivo. The aim of the present study was to systematically explore RNAi-mediated gene-silencing of known T cell-specific model signaling molecules in primary murine T cells in vitro and in vivo. We demonstrate that siRNA delivery and subsequent silencing of T cell specific genes is substantially increased, if murine T cells were activated prior siRNA transfection. Silencing of ZAP70, p56Lck as well as PLC-γ1 protein expression resulted in impaired function of T cells in vitro. Furthermore, delayed type hypersensitivity (DTH) was ameliorated in vivo after adoptive transfer of ZAP70-silenced T cells. The combination of RNAi-mediated gene silencing and adoptive transfer of gene-silenced T cells, thus, may allow the identification and analysis of T cell-specific targets for therapeutic intervention. Additionally, this model system may represent an alternative to conventional time consuming and cost intensive gene targeting approaches.