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 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 mechanism 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 act through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

RNA-Seq Analysis of Activated PBMC Treated With LMWF5A Predicts an Anti-Inflammatory Mode of Action and Similar Drug Targets to Dexamethasone

“…Similarly, LMWF5A has also been proven to modulate transcription factor activity to decrease pro-in ammatory cytokine levels. Its ability to reduce TNF and IL-1β release has been linked to its effects on the NFκB-repressing PPARγ and AhR pathways in LPSstimulated PBMC, and it has also been shown to prevent NFκB reporter activity [9]. PTGER4 (or EP4) is a transmembrane, G-coupled protein receptor that becomes activated when bound to the cyclooxygenase (COX) pathway product PGE2, which is induced during in ammation.…”

“…LPS was used in two of the three conditions in this study to stimulate an immune response in PBMC, and its ranking as the most negatively correlated upstream regulator to LMWF5A highlights the fact that LMWF5A strongly counteracts TLR4-mediated in ammation. Moreover, the antiin ammatory effects of LMWF5A on cytokine release and transcription factor activity have been extensively studied using cells treated with LPS as a TLR4 stimulant [7,9,48]. poly rI:rC-RNA is a dsRNA mimic that can simulate infection with a dsRNA virus and activate TLR3 [49], suggesting that LMWF5A may also offset the actions of other TLR-driven pathways; the relationships between LMWF5A and other TLRs are currently under investigation.…”

“…Upon recognition of IFNs, a variety of interleukins, or other cytokines, STAT1 is phosphorylated, mainly by JAK kinases, and activated to drive a pro-in ammatory cascade [52]. LMWF5A was also previously demonstrated to inhibit the ability of STAT1 to bind its cognate DNA sequence in LPS-stimulated PBMC, implicating regulation of this transcription factor as part of the LMWF5A mode of action [9]. Inhibition of the JAK-STAT pathway is an effective therapeutic strategy for the treatment of in ammatory conditions, including rheumatoid arthritis (RA), psoriasis, in ammatory bowel disease [53] as well as COVID-19 [54].…”

“…Much research on the biologic drug LMWF5A has focused on its ability to inhibit release of the pro-in ammatory cytokine tumor necrosis factor (TNF) from immune cells, but LMWF5A has also been shown to affect interleukin (IL)-1β, IL-6, IL-10, IL-12, interferon (IFN) γ, chemokine (C-X-C motif) ligand (CXCL) 9, CXCL10, CXCL11, prostaglandin (PG) E2, PGD2, and 15d-PGJ2 due to its impact on regulators upstream of in ammatory protein production [4][5][6][7][8][9][10]. Known mechanisms by which LMWF5A exerts its immunomodulatory effects include decreasing pro-in ammatory transcription factor activity (nuclear factor κB [NFκB] and signal transducer and activator of transcription [STAT]) [9], increasing antiin ammatory transcription factor activity (peroxisome proliferator-activated receptor [PPARγ] and aryl hydrocarbon receptor [AhR]) [7,9], and reducing endothelial cell permeability [11]. These modes of action predict that LMWF5A attenuates in ammation, promotes the resolution of immune responses, and restores homeostasis, supporting its use in the treatment of various in ammatory conditions.…”

AMP5A modulates Toll-like receptors 7 and 8 single-stranded RNA immune responses in PMA-differentiated THP-1 and PBMC

“…For instance, AMP5A demonstrates an ability to reduce the secretion of proinflammatory cytokines and chemokines associated with cytokine storms (i.e. IL-1β, IL-6, IL-12, TNFα, and CXCL10) from immune cells activated through TLRs and/or T-cells stimulated via the T-cell receptor [18][19][20][21][22][23]. Importantly, AMP5A inhibits nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB), based on reduced p65 and RelB subunit DNA binding activity, in lipopolysaccharide(LPS)-stimulated peripheral blood mononuclear cells (PBMC) [23].…”