Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases

RORγt and RORα signature genes in human Th17 cells

<div><p>RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.</p></div

Upstream regulators of the 24 genes down-regulated by RORγt siRNAs or inverse agonists in human Th17 cells were predicted from IPA analysis and the top 20 regulators are shown.

<p>These regulators were ranked by their P values which reflect the enrichment of the 24 genes regulated directly or indirectly by these upstream regulators. The activation status of upstream regulators was determined from the treatment effect on the involved genes and presented as activation z-scores, with positive value for activated status, and negative value for inhibited status.</p

Expression of RORγt and RORα4 in human CD4 T cells during Th17 differentiation.

<p>Expression of RORγt isoform from the RORC gene (A) and RORα4 isoform from the RORA gene (B) was determined from detection of isoform specific exon sequences in RNA-Seq data analysis. Exons utilized by different isoforms of the RORC and RORA genes are shown in green, and the frequency of exon sequences in RNA-Seq data are shown in red in the histograms. DMSO treated human CD4 T cell samples from 2 donors at different time points of activation under Th17 differentiation conditions were used for isoform expression analysis.</p

A total of 24 genes were down-regulated by RORγt siRNAs or inverse agonists in human Th17 cells.

<p>The change in gene expression resulted from treatment of RORγt siRNAs or RORγt inverse agonists was presented in fold change of the geomean average calculated from samples of the two donors.</p

Global gene expression profile with RNA-Seq identified genes differentially regulated by RORγt or RORα siRNA in human CD4 T cells during Th17 differentiation.

<p>RNA sequencing profile was performed on RNA samples extracted from human CD4 T cells treated with RORγt or RORα siRNA, or RORγt inverse agonists as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181868#pone.0181868.s001" target="_blank">S1 Table</a>. Genes down-regulated by RORγt (A) and RORα (B) siRNAs, or up-regulated by RORγt or RORα siRNAs (C) were identified in analysis of RNA-Seq data using the criteria described in Materials and Methods. Each column represents log2 ratios of selected genes in one of the 72 comparisons, including comparison of untreated Th17 cells with RORγt or RORα siRNA transfected cells or RORγt inverse agonist treated cells at two time points and T cells from 2 independent donors.</p

RORγt or RORα siRNA effect on IL-17A transcript in human CD4 T cells during Th17 differentiation.

<p>Human CD4 T cells purified from 2 donors were activated under Th17 differentiation condition as described in Material and Methods. RORγt and RORα siRNAs, as well as control siRNAs were transfected into CD4 T cells prior to Th17 differentiation and samples were collected at different time points as indicated. Expression of RORγt (A), RORα (B) and IL-17A (C) transcripts was measured by RT-PCR. IL-17A cytokine released in culture supernatants (D) was measured by ELISA. Statistical significance of the difference between RORC or RORA siRNA treated samples compared to mock samples at different time points was analyzed with 2-way ANOVA and P values were presented as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.</p

IL-17A transcript in human CD4 T cells reduced by RORγt siRNA or inverse agonist at later but not early stage of Th17 differentiation.

<p>Human CD4 T cells were activated under Th17 differentiation conditions as described in Material and Methods. RORγt siRNAs (RORC siRNA_2 & 4) and control scramble siRNA (control_3) were transfected into CD4 T cells prior to Th17 differentiation and samples were collected at different time points and the expression of RORγt (A) and IL-17A (B) transcripts was measured by RT-PCR as described in Materials and Methods. RORγt compound D at 0.1 and 0.5 μM and DMSO vehicle control were tested in a similar manner in Th17 differentiation. RORγt (C) and IL-17A (D) transcripts were measured by RT-PCR at different time points. Statistical significance of RORC siRNA or compound treated samples in reducing RORγt or IL-17A mRNA at different time points was analyzed with 2-way ANOVA and P values were indicated as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.</p

Top 10 canonical pathways in IPA associated with the 24 genes that were down-regulated by RORγt siRNAs or inverse agonists in human Th17 cells.

<p>The top 10 canonical pathways were selected by the p-value of overlap of the 24 genes and the genes in each pathway.</p