Abstract
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.
Highlights
Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases
LMWF5A suppresses distinct pro‐inflammatory cytokines in LPS‐stimulated peripheral blood mononuclear cells (PBMC) We have previously shown that LMWF5A exhibits antiinflammatory properties with an ≈35% inhibition of Tumor necrosis factor alpha (TNFα) from LPS-induced human ex vivo PBMC cultures [22]
(0.58 ± 0.08-fold change), Interleukin 1 beta (IL-1β) (0.71 ± 0.05-fold change), and Interleukin 12 (IL-12) (0.34 ± 0.07-fold change) (Table 1). These findings demonstrate that in addition to TNFα, LMWF5A treatment of LPS-stimulated PBMC results in a distinct cytokine signature, with reduced release of specific proinflammatory signals after 24 h in culture
Summary
Dysregulation 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. Dysregulation of transcription and the expression of inflammatory proteins has been implicated in the pathogenesis of a variety of chronic diseases including arthritis, atherosclerosis, diabetes, pulmonary fibrosis, kidney disease, and inflammatory bowel disease [1]. These conditions exhibit a range of clinical symptoms, they all involve an overactivation of the immune response and/or an inability of the immune response to progress towards resolution and healing. Chronic inflammation can manifest under conditions where pro-inflammatory programs sustain the production of cytokine and/or resolving programs fail to eventuate [4]
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