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Downregulation of Inflammatory Cytokine Release from IL-1? and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

ABSTRACT: The inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. Myeloid differentiation primary response 88 (MyD88) is an essential protein recruited after lipopolysaccharide (LPS) and interleukin (IL)-1? stimulation, a process that converges in nuclear factor kappa B (NF-?B) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. The inhibition of MyD88 has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. In this study, we investigate the effect of MyD88 dimerisation inhibitor ST2825 on cytokine production from rhIL-1? and LPS-stimulated peripheral blood mononuclear cells (PBMC) from healthy blood donors (HBD). ST2825 significantly downregulates the production of IFN-?, IL-6, IL-12, IL-2, IL-15, IL-7, VEGF, IL-1Ra, IL-4, IL-5, IL-13 and IL-9 (p < 0.05) in LPS-stimulated PBMC. Moreover, ST2825 had a relatively low impact on IL-1? signalling pathway inhibition, showing that only a few specific cytokines, such as IFN-? and IL-1Ra, are inhibited in rhIL-1?-stimulated PBMC (p < 0.01). In conclusion, MyD88 dimerisation inhibitor ST2825 showed high efficacy by inhibiting pro- and anti-inflammatory cytokine production in LPS-stimulated PBMC. Moreover, although rhIL-1? induced a sustained cytokine production (p < 0.05), ST2825 did not show a significant effect in the secretion of neither pro- nor anti-inflammatory cytokines in rhIL-1?-stimulated PBMC.

SUBMITTER: Ramirez-Perez S

PROVIDER: S-EPMC7570868 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Downregulation of Inflammatory Cytokine Release from IL-1β and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

Ramírez-Pérez Sergio S Hernández-Palma Luis Alexis LA Oregon-Romero Edith E Anaya-Macías Brian Uriel BU García-Arellano Samuel S González-Estevez Guillermo G Muñoz-Valle José Francisco JF

Molecules (Basel, Switzerland) 20200921 18

The inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. Myeloid differentiation primary response 88 (MyD88) is an essential protein recruited after lipopolysaccharide (LPS) and interleukin (IL)-1β stimulation, a process that converges in nuclear factor kappa B (NF-κB) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. The inhibition of MyD88 has shown efficacy by decrease inflammatory respon ...[more]

PMID: 32967164

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Project description:BackgroundDysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-β, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release.MethodsELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1β relative potency were then employed to confirm the involvement of enriched pathways and TFs.ResultsLMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1β, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1β release.ConclusionIn this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

Project description:Treatment of PBMC with 0.6 pM cytokine from 0-24h Abstract: Background Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. Results: We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons a, b, w and g, IL12 and TNFa; and (2) characterise the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNg stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNg and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFa stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNg, and emergent properties associated with IFN-mediated activation of mixed cell populations. Conclusions: This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the detection and definition of host immune responses in a variety of disease settings. PBMC extraction and stimulation Human primary peripheral blood mononuclear cells (PBMCs) were purified from whole blood of healthy donors using Ficoll-Paque PLUS (GE Healthcare) according to manufacturers instructions. PBMCs were incubated at 1.5-2.0 x 106 cells/well for 24 h before stimulation in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum and 2 mM L-glutamine (Invitrogen) at 37C, 5% CO2. Cells were treated with 0.006 pM, 0.6 pM or 60 pM recombinant IFNg (R&D Systems), and sampled at time intervals from 0.5 h to 12 h after stimulation. Additionally, cells were treated with 0.1 % BSA/PBS alone and used for untreated (mock) control time courses. RNA extraction and amplification Total RNA was extracted in TRIzol LS (Invitrogen) followed by standard chloroform purification and isopropanol precipitation. RNA was resuspended in RNase-free water, quantitated on the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and stored at -80C. 500 ng total RNA was amplified using the MessageAmp modified Eberwine linear amplification procedure (Applied Biosystems). All samples to be compared were extracted and amplified together. Microarray analysis 4 ug amplified RNA was labelled with Cy5-dUTP (GE Healthcare) and combined with 3 ug of Cy3-labelled reference cDNA derived from a pool of RNA derived from a panel of 11 human cell lines (Stratagene Universal Human Reference RNA). The samples were washed and concentrated using MinElute columns (Qiagen) and competitively hybridised to custom printed cDNA microarrays containing 37,632 elements for approximately 18,000 unique human genes. The hybridised slides were scanned using a GenePix 4000A microarray scanner (Axon Instruments). Comparative spot intensities were calculated from the images, and areas of poor quality excluded from further analysis using GenePix Pro 6.0 (Axon Instruments). Data were deposited in the Stanford Microarray Database. Analysis was restricted to cDNA elements with a regression correlation of > 0.6, fluorescence intensities of > 2.5 fold signal/background in Cy3 or Cy5 channels and a minimum signal intensity of > 100 in both channels for at least 80% of the arrays. The expression ratios were normalised for array variation, and the data zero-transformed using a custom-designed Microsoft Excel macro (C. Liu, Stanford). The statistical package SAM (Significance Analysis of Microarrays, version 1.15) was used to identify genes significantly differentially expressed in the normalised data sets by pairwise comparison with a minimum 2 fold cutoff at a false discovery rate of < 1% of the median. The transformed datasets were then hierarchically clustered using Cluster 2.11 and the results displayed using Treeview 1.60. A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed.

Dataset Information

Downregulation of Inflammatory Cytokine Release from IL-1? and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

Publications

Downregulation of Inflammatory Cytokine Release from IL-1β and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

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