LMWF5A suppresses cytokine release by modulating select inflammatory transcription factor activity in stimulated PBMC

Abstract

Background 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. 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. Methods ELISA 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. Results LMWF5A 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. Conclusion In 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.

A Randomized Controlled Trial to Evaluate the Safety and Efficacy of a Novel Inhaled Biologic Therapeutic in Adults with Respiratory Distress Secondary to COVID-19 Infection

“…In patients who develop respiratory distress associated with COVID-19, the activation of the innate immune system leads to a dysregulated or ‘hyper-inflammatory’ response, resulting in the excess release of innate pro-inflammatory cytokines by alveolar macrophages and neutrophils as part of a “cytokine storm.” The severity of COVID-19 and related inflammatory complications, such as ARDS, is closely associated with increased serum levels of pro-inflammatory cytokines, including TNFα, IL-1β, IL-6, IL-12, and CXCL-10, and accompanied by a corresponding decrease in anti-inflammatory cytokines [ 5 , 29 , 30 ]. In vitro studies using AMP5A show it can modulate these cytokine levels in various immune cell models; this activity is suggested to decrease inflammation and promote the resolution of the immune response [ 11 , 31 , 32 ], particularly in the treatment of the hyperinflammatory response related to COVID-19 [ 26 ].…”

“…AMP5A is a novel non-steroidal, anti-inflammatory biologic agent containing a cyclized dipeptide and other small molecules with immunomodulatory and anti-inflammatory properties. In vitro experiments in activated immune cells show that AMP5A inhibits the production of inflammatory cytokines, including TNFα, IFNγ, IL-1β, IL-6, IL-12, and CXCL-10, by interfering with the inflammatory transcription factor NF-κB while at the same time activating immunomodulatory transcription factors aryl hydrocarbon receptor (AhR) and peroxisome proliferator activator receptor gamma (PPARg) [ 11 ]. AMP5A is also in clinical development as an anti-inflammatory therapy for osteoarthritis and has been safely administered as an intra-articular injection for the reduction of pain and improvement in function in patients with osteoarthritis of the knee [ 12 , 13 ].…”