John Mo

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague–Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.

Specific accumulation of orally administered redox nanotherapeutics in the inflamed colon reducing inflammation with dose-response efficacy

Although current medications for ulcerative colitis (UC) are effective to some extent, there are still some limitation of their use due to the non-specific distribution, drug metabolism in the gastrointestinal tract, and severe adverse effects. In our previous studies, we developed oral redox nanoparticles (RNP(O)) that specifically accumulated and scavenged overproduced reactive oxygen species (ROS) in an inflamed colon. However, the mechanism leading to specific accumulation of RNP(O) in an inflamed colon is still unclear. In this study, we investigated the cellular uptake of RNP(O) into ROS-treated epithelial colonic cells in vitro, and compared to the untreated cells, found a significantly increased uptake in ROS-treated cells. In vivo, we discovered that orally administered RNP(O) were not internalized into the cells of a normal colon. A significant amount of disintegrated RNP(O) was detected in the cells of an inflamed colon of dextran sodium sulfate (DSS)-induced colitis mice, resulting in scavenging of ROS and suppression of inflammation with low adverse effects. Furthermore, we confirmed a significant reduction of disease activity and a robust dose response efficacy following RNP(O) treatment in acute DSS-induced colitis mice, outperforming the positive control 5-aminosalicylic acid. Oral administration of RNP(O) is a promising approach to develop a new therapy for UC disease.

Dipeptidyl Peptidase 1 Inhibitor AZD7986 Induces a Sustained, Exposure‐Dependent Reduction in Neutrophil Elastase Activity in Healthy Subjects

Neutrophil serine proteases (NSPs), such as neutrophil elastase (NE), are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. High NSP levels can be detrimental, particularly in lung tissue, and inhibition of NSPs is therefore an interesting therapeutic opportunity in multiple lung diseases, including chronic obstructive pulmonary disease (COPD) and bronchiectasis. We conducted a randomized, placebo‐controlled, first‐in‐human study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple oral doses of the DPP1 inhibitor AZD7986 in healthy subjects. Pharmacokinetic and pharmacodynamic data were analyzed using nonlinear mixed effects modeling and showed that AZD7986 inhibits whole blood NE activity in an exposure‐dependent, indirect manner—consistent with in vitro and preclinical predictions. Several dose‐dependent, possibly DPP1‐related, nonserious skin findings were observed, but these were not considered to prevent further clinical development. Overall, the study results provided confidence to progress AZD7986 to phase II and supported selection of a clinically relevant dose.

The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

Objectives Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ+ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. Results In resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. Conclusions T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.

Cytokine production by activated plasmacytoid dendritic cells and natural killer cells is suppressed by an IRAK4 inhibitor

BackgroundIn systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ).MethodsPlasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ.ResultsIn healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74–95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) < 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes).ConclusionsThe IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE.

FRI0257 The stat4 sle risk allele rs7574865[t] is associated with increased il-12-induced ifn-gamma production in t cells from sle patients

Background Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. Objectives This study aimed to clarify how the SLE-associated intronic STAT4 risk variant rs7574865[T] affects the function of immune cells in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 52 SLE patients in remission (SLEDAI-2K≤4). STAT4 and STAT1 protein levels and phosphorylation status in response to interferon (IFN)-α, IFN-γ, or interleukin (IL)−12 were determined, before and after pre-activation of cells with phytohaemagglutinin (PHA) and IL-2, in CD56dim NK cells, CD56bright NK cells, B cells, CD4+ T cells, CD8+ T cells and monocytes by flow cytometry. The frequency of IFN-γ+ cells upon IL-12 or PMA (phorbol 12-myristate-13-acetate) stimulation and the frequency of T-bet+ cells was determined in PHA/IL-2 pre-activated cells. Cellular responses and phenotypes were correlated with STAT4 risk allele carriership (rs7574865[T], 21 homozygous protective, 22 heterozygous and 9 homozygous risk) using an additive linear regression model. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i, Compound 35) or JAK2 (JAK2i, BMS-911543) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. Results In resting PBMCs, the STAT4 risk allele was neither associated with protein levels of STAT4 or STAT1, nor cytokine-induced phosphorylation of STAT4 (pSTAT4) or STAT1 (pSTAT1). However, following PHA/IL-2-activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 (p=0.04), resulting in increased pSTAT4 in response to IL-12 (p=0.003) and IFN-α (p=0.04). Analysis of T cell subsets revealed that the effect was seen in CD45RA+CD57– naïve and CD45RA–CD57– memory CD8+ T cells, but not in CD45RA–CD57+ memory or CD45RA+CD57+ effector CD8+ T cells. A slight increase in STAT4 protein levels and IL-12-induced pSTAT4 was also observed in CD4+ T cells from STAT4 risk allele carriers (p=0.08 and p=0.09, respectively). STAT4 risk allele carriers displayed an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells (p=0.03 for both), whereas PMA-induced IL-12 production was normal (p=0.31 and p=0.10, respectively). T-bet expression was not correlated to the STAT4 genotype. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. Conclusions T cells from SLE patients carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment. Disclosure of Interest N. Hagberg: None declared, M. Joelsson: None declared, D. Leonard: None declared, S. Reid: None declared, M.-L. Eloranta: None declared, J. Mo Employee of: AstraZeneca, M. Nilsson Employee of: AstraZeneca, A.-C. Syvänen: None declared, Y. Bryceson: None declared, L. Rönnblom: None declared

Simple and robust two-step ex vivo whole blood stimulation assay suitable for investigating IL-17 pathway in a clinical laboratory setting

We describe a simple and robust tube-based ex vivo whole blood stimulation procedure suitable for use in clinical laboratories by multiple operators on repeated occasions to study cytokine production in phase 1 human trials of investigational medicines, developed by the authors specifically to study IL-17A expression in man. The stimulation procedure is further proposed as a useful tool for biomarker assay development and validation, for example to prepare quality control samples without reliance on recombinant materials for spiking.

THU0247 The Effect of Hydroxychloroquine and IRAK4 Inhibition on The IFN-A and TNF-A Production Induced by Sle Related Immune Complexes

Background Patients with systemic lupus erythematosus (SLE) have an ongoing interferon (IFN)-α production due to the stimulation of Toll-like receptors (TLR) 7 and 9 in plasmacytoid dendritic cells (pDCs) by immune complexes (ICs) containing self-derived nucleic acid. Such interferogenic ICs also trigger the production of TNF-α by several immune cells. Produced TNF-α in SLE contributes to organ inflammation such as nephritis and arthritis, but can also modulate the central autoimmune process as demonstrated by the induction of a lupus-like syndrome after anti- TNF-α treatment. Objectives To clarify the effect on IFN-α and TNF-α production by IC-stimulated immune cells from healthy individuals and SLE patients exposed to hydroxychloroquine (HCQ) and an IRAK4 inhibitor Methods pDC and NK cells were isolated from peripheral blood of healthy individuals and monocyte depleted PBMCs from SLE patients. Cells were stimulated with RNA-containing ICs (RNA-IC) comprised of SLE-IgG and U1snRNP particles in the presence or absence of HCQ, which blocks endosomal TLR7/9 activation, or a IL-1 receptor associated kinase (IRAK) 4 inhibitor (AZ13732754–004) acting downstream of TLR 7/9. IFN-α and TNF-α production was measured after 20 h by immunoassays or by intracellular staining using flow cytometry. Results RNA-IC induced IFN-α production selectively in pDC, whereas TNF-α was produced by both pDC and NK cells. Co-cultivation of pDC and NK cells enhanced the production of IFN-α (28-fold) and TNF-α (15-fold). TNF-α was rapidly produced by NK cells (5 h), but exhibited a delayed production in pDC (9 h). All IFN-α positive pDCs (13.6%) were TNF-α positive, whereas a fraction of pDCs (7.1%) were only TNF-α positive. HCQ completely inhibited the IFN-α and TNF-α production by pDCs, but did not affect TNF-α production by NK cells. Preliminary studies showed that cells from HCQ-treated SLE patients produced little IFN-α (mean 3 U/ml vs 27 U/ml in not HCQ-treated) in response to RNA-IC [NH1], whereas TNF-α production was maintained (mean 760 U/ml [NH2] vs 407 U/ml in not HCQ-treated). Inhibition of IRAK4 effectively blocked IFN-α production in both pDC (94%) and pDC-NK cell co-cultures (91%). Conclusions The IFN-α production induced by RNA-IC is completely blocked by HCQ and the IRAK4 inhibitor AZ13732754–004, suggesting that administration of an IRAK4 inhibitor could be an effective treatment strategy in SLE. The observation that HCQ inhibited the pDC derived TNF-α production, but not TNF-α released from other immune cells in SLE patients, may explain why HCQ treatment is only partially effective in SLE. In addition, our observation indicates that RNA-IC stimulated TNF-α production is induced by different mechanisms in pDC and NK cells. Further investigations are needed to clarify if IRAK4 inhibition has a broader inhibitory effect than HCQ on the production of proinflammatory cytokines, and if inhibition of IRAK4 should be explored as a therapeutic option in SLE. Disclosure of Interest K. Hjorton Grant/research support from: Study partly financed by Astra Zeneca, N. Hagberg: None declared, O. Berggren: None declared, J. Mo: None declared, J. Sandling: None declared, M.-L. Eloranta: None declared, L. Rönnblom: None declared