Lack of evidence for expression and function of IL-39 in human immune cells.

Members of the IL-6/IL-12 cytokine family are critical regulators of innate and adaptive immunity and have emerged as key players controlling inflammatory and autoimmune disorders. This cytokine family comprises of IL-12, IL-23, IL-27, and IL-35, each consisting of distinct α- and ß-cytokine subunits that form heterodimers. A new member of this family, IL-39, was identified in the murine species and was shown to consist of the IL-23p19 and Epstein-Barr Virus-induced 3 (EBI3) subunits. Subsequently, it was shown that IL-39 was implicated in the immunopathogenesis of murine experimental lupus erythematosus. The existence of IL-39 in the human system has yet to be confirmed. Based on the clinical success of IL-23p19 neutralizing approaches in moderate-to-severe psoriasis, anti-IL-23p19 antibodies in the clinic may not only neutralize IL-23, but additionally IL-39, implying that IL-39 might also contribute to the pathogenesis of psoriasis. It is therefore pivotal to demonstrate IL-39 expression and to characterize its function in the human system. In this study, we provided evidence for the existence of secreted heterodimeric p19 and EBI3 complexes in supernatants originating from p19 and EBI3 transfected HEK293FT cells. We attempted to detect IL-39 expression from stimulated human primary B cells, human keratinocytes and in vitro polarized human macrophages. Whereas, the expression of p19 and EBI3 mRNA was elevated, we failed to detect p19 and EBI3 heterodimers. Functional assays were conducted with conditioned media containing human IL-39 or with a human recombinant IL-39 Fc protein. Immune cells targeted by IL-39 in mouse, such as neutrophils and PBMCs, did not respond to human IL-39 stimulation and IL-39 failed to activate STAT3 in a reporter cell line. These results suggest that, while the secretion of p19/EBI3 complexes can be forced in human cells, it is secreted below the lower quantity of detection or it has no functional role.

Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses.

The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the in vitro pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as IL17F, IL22, IL26, IL23R, and CCR6. The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORß. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in ex vivo immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human ß defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies.

Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo.

Retinoic acid receptor-related-orphan-receptor-C (RORγt) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of RORγt in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in vitro and in vivo pharmacology of a potent and selective small-molecular-weight RORγt inverse agonist. The compound binds to the ligand binding domain (LBD) of RORγt leading to displacement of a co-activator peptide. We show for the first time that a RORγt inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and γδT-cells and attenuated transcription of RORγt target genes. The inhibitor showed good in vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex vivo recall assays. In summary, we demonstrate that inhibiting RORγt by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.