Maryanne L. Brown

Protein Kinase C-θ Mediates Negative Feedback on Regulatory T Cell Function

Yin-Yang T Cell Signaling Immune responses are kept in check by CD4+ regulatory T cells (Treg) that suppress other immune cells, including CD4+ effector T cells (Teff). Treg and Teff cells have many signaling components in common, yet triggering through their T cell receptors (TCRs) leads to very different outcomes. Zanin-Zhorov et al. (p. 372, published online 25 March) compared the recruitment of signaling molecules to the immunological synapse after TCR triggering in Treg and Teff cells. Although Treg cells do form synapses, signaling molecules that promote Teff activation, such as protein kinase C-θ (PKC-θ), were not recruited. Inhibition or depletion of PKC-θ in Treg cells led to suppressive activity against Teff cells, whereas costimulation enhanced PKC-θ recruitment and less suppression. Together, this suggests that PKC-θ is inflammatory in both Treg and Teff cells; however, by excluding it from the synapse, Treg cells are able to maintain suppression in the face of TCR signaling. Suppressive T cells repurpose inflammatory signaling pathways to promote their suppressive functions. T cell receptor (TCR)–dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor–α (TNF-α). Protein kinase C-θ (PKC-θ) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-θ was sequestered away from the Treg immunological synapse. Furthermore, PKC-θ blockade enhanced Treg function, demonstrating PKC-θ inhibits Treg-mediated suppression. Inhibition of PKC-θ protected Treg from inactivation by TNF-α, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-θ–mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases.

Circulating Monocytes Are Reduced by Sphingosine-1-Phosphate Receptor Modulators Independently of S1P3

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.

Pathogenic mechanisms and experimental models of multiple sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disease that affects more than 1 million people worldwide and severely compromises motor and sensory function through demyelination and axonal loss. This review covers current therapies, lessons learned from failed clinical trials, genetic susceptibility, key cell types involved, animal models, gene expression, and biomarker information. The current first-line therapies for MS include the type I interferons (IFN-I) and glatiramer acetate (GA) but because of their limited effectiveness new therapeutic modalities are required. Tysabri is an anti very late antigen-4 antibody that antagonizes the migration of multiple cell types and appears more efficacious as compared to the IFNs or GA. Tysabri blocks the transmigration of T cells and monocytes, which indicates that blocking multiple cell types may increase the effectiveness of the therapy. However, this therapy may increase the risk of progressive multifocal leukoencephalopathy. The major cell types hypothesized to be pathogenic include T cells and antigen-presenting cells, including B cells. The correlation of the animal model experimental autoimmune encephalomyelitis (EAE) of MS and its predictive value to determine efficacy in the clinic appears limited. However, all current therapies do demonstrate efficacy in EAE models. There are also examples of mechanisms that have worked in EAE but have failed in the clinic, such as the TNFα antagonists and anti-p40 (a subunit of IL-12 and IL-23). The MS field would benefit if clinical biomarkers were available to monitor clinical efficacy. The etiology of MS remains elusive but additional understanding of mechanisms involved in the pathogenesis of MS may guide us to more effective treatment and management of this autoimmune disease.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

Involvement of GATA3 in Protein Kinase C θ-induced Th2 cytokine expression

Protein kinase C θ (PKCθ) is essential for T cell activation, as it is required for the activation of NF‐κB and expression of IL‐2. PKCθ has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCθ in the regulation of T helper cells, we used PKCθ‐deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCθ produced significantly less TNF‐α and IL‐2. The expression of Th2 cytokines, including IL‐4, IL‐5, IL‐10, IL‐13 and IL‐24 was significantly reduced in PKCθ‐deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCθ‐deficient T cells. Overexpression of GATA3 by retroviral infection in PKCθ‐deficient T cells resulted in increased expansion of IL‐4‐producing T cells and higher IL‐4 production than that of wild type Th2 cells. IL‐5, IL‐10, IL‐13 and IL‐24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCθ regulates Th2 cytokine expression via GATA3.

CCR1 Plays a Critical Role in Modulating Pain through Hematopoietic and Non-Hematopoietic Cells

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freunds adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

02.50 Intestinal overexpression of human tnf triggers sacro-iliitis

An intriguing link exists between gut and joint inflammation in spondyloarthritis (SpA). About 50% of patients has subclinical (eg, microscopic) gut inflammation, which represents a risk factor for development of Crohn’s disease, sacroiliac inflammation and evolution in to ankylosing spondylitis. However, the underlying mechanisms are still relatively poorly understood. Our goal was to examine the relationship between TNF, microscopic gut inflammation and axial inflammation. Therefore, we examined in situ expression of TNF, TNFR1 and TNFR2 using triple in situ hybridisation in gut biopsies of human SpA patients and found marked upregulation of TNF in inflamed versus non-inflamed biopsies. We also noted a predominant upregulation of TNFR1 on intestinal epithelium and TNFR2 in lamina propria respectively. Of interest, IL-17 and IL-23 were also markedly increased while IL-22 was most abundant in chronically inflamed samples. In line with this, we found that patients with gut inflammation had a higher need for anti-TNF therapy and their degree of clinical response after anti-TNF was also markedly higher. We speculated that TNF in the gut represents an important risk factor for disease severity and progression in SpA. To investigate this further we generated intestinal specific human TNF transgenic mice, in which hTNF is under control of a rat iFABP (fatty acid binding protein) promoter, generating a mouse-model over-expressing human TNF in the ileum. These mice, together with wild type littermates, were evaluated for the development of arthritis up until the age of 13 weeks after which they were euthanized and ankle and sacroiliac joints as well as ileum were processed for histology. Transgenic mice exhibit a runt phenotype and hallmarks of inflammatory bowel disease, including increased intestinal permeability and inflammation compared to their wild-type littermates. While in peripheral joints no clear signs of arthritis were observed, the sacroiliac joints in transgenic mice, by contrast, showed marked signs of inflammation as well as bone erosion and destruction. These data propose a new paradigm that gut-derived TNF is sufficient to trigger sacroiliitis and provide an alternate explanation on the relationship between gut inflammation, evolution to inflammatory bowel disease and axial inflammation in SpA.

THU0128 Evaluation of Safety, Pharmacokinetics and Pharmacodynamics of BI 638683, A Novel CCR1 Antagonist

Background Monocytes and macrophages play a major role in the pathogenesis of rheumatoid arthritis (RA). The chemokine receptor CCR1 regulates migration of these cells to synovial tissue and the bone. It is expected that sustained full antagonism of CCR1 will block trafficking of monocytes to the site of inflammation and will block the upregulation of cytokines, adhesion molecules and MMPs to slow the progression of joint damage in RA. Objectives This clinical study investigated safety, tolerability, pharmacokinetics and pharmacodynamics of the novel selective CCR1 antagonist BI 638683. Methods In a randomised, blinded, placebo-controlled trial, single rising doses from 1 to 700 mg BI 638683 or placebo were administered to 63 young healthy male volunteers (mean age 32 ± 9 years; mean BMI was 24.7 ± 2.4 kg/m2). Subjects were randomised in a 6:2 ratio to active or placebo per group. Two distinct mechanistic blood-based biomarker assays were used to establish a PK/PD relationship: 1. inhibition of MIP1α-induced CCR1 receptor internalization (RI) and 2. inhibition of RANTES-induced CCR1 dependent gene expression of CCL2, CLEC5A, RAB7B and PPARG Results All doses of BI 638683 were well tolerated. There were no serious adverse events (AE) and no AEs of severe intensity. There was no dose-relationship of AEs and no difference in the number or category of reported AEs in subjects treated with BI 638683 (7/47; 14.9%) compared to those treated with placebo (3/16; 18.8%). The most frequent AEs were GI disorders which were reported in 5/47 (10.6%) subjects treated with BI 638683 and 2/16 (12.5%) subjects treated with placebo. Only one event of nausea in the 700g mg dose group was rated as drug related. Plasma exposure of BI 638683 increased in a near dose-linear fashion, with a terminal t½ of 5-15 h. Two hours after dosing (the pharmacokinetic tmax), a dose of 150 mg BI 638683 inhibited RI by approximately 90%. The percentage of inhibition was directly related to the dose of BI 638683. At the 700 mg dose, an 83% inhibition of RI was measured at 24 h after dosing. Maximal inhibition of mRNA expression of the 4 CCR1 dependent marker genes was reached with a dose of 75 mg BI 638683 at the tmax. 24 h after dosing, ≥90% mean inhibition was still maintained for CCL2 and PPARG mRNAs by doses of 300 mg and higher, and for RAB7B mRNA by doses of 500 mg and higher. For CLEC5A, inhibition of 84% and 91% was achieved for the 500 and 700 mg dose, respectively. Conclusions Treatment with BI 636683 was well tolerated. The biomarker assays indicated substantial inhibition and demonstrated proof of mechanism for BI 638683 as a CCR1 inhibitor in early stage of clinical development, and the PK/PD data using these biomarkers can be used to determine appropriate dosing for this compound. Disclosure of Interest : P. Baum Employee of: Boehringer-Ingelheim, C. Schoelch Employee of: Boehringer-Ingelheim, H. Zimdahl-Gelling Employee of: Boehringer-Ingelheim, M. Brown Employee of: Boehringer-Ingelheim, D. Webb Employee of: Boehringer-Ingelheim, S. Padula Employee of: Boehringer-Ingelheim, J. Hilbert Employee of: Boehringer-Ingelheim, T. Giessmann Employee of: Boehringer-Ingelheim, J. Steffgen Employee of: Boehringer-Ingelheim DOI 10.1136/annrheumdis-2014-eular.2352