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

Background: Dysregulation of antiviral immunity has been implicated in the progression of acute respiratory syndrome coronavirus 2 infection into severe cases of coronavirus disease of 2019 (COVID-19). Imbalances in the inflammatory response drive the overabundant production of pro-inflammatory cytokines and chemokines. The low molecular weight fraction of 5% human serum albumin commercial preparation (AMP5A) is a novel biologic drug currently under clinical investigation for the treatment of osteoarthritis and the hyperinflammatory response associated with COVID-19. This study aims to elucidate AMP5A effects following the activation of immune cells with agonists of Toll-like receptor (TLR) 7 and/or 8, which detect ssRNA viral sequences. Methods: CXCL10 ELISAs were used to evaluate the dynamics of myeloid cells activated with CL075 and CL307, agonists of TLR7/8 and TLR7, respectively. In addition, enrichment analysis of gene sets generated by ELISA arrays was utilized to gain insight into the biologic processes underlying the identified differentially expressed cytokine profiles. Finally, relative potency (REP) was employed to confirm the involvement of mechanisms of action paramount to AMP5A activity. Results: AMP5A inhibits the release of CXCL10 from both CL075- and CL307-activated PMA-differentiated THP-1 and peripheral blood mononuclear cells. Furthermore, AMP5A suppresses a distinct set of pro-inflammatory cytokines (including IL-1ß, IL-6, IL-12, and CXCL10) associated with COVID-19 and pro-inflammatory NF-κB activation. REP experiments using antagonists specific for the immunomodulatory transcription factors, peroxisome proliferator-activated receptor γ, and aryl hydrocarbon receptor, also indicate that these pathways are involved in the ability of AMP5A to inhibit CXCL10 release. Conclusion: Due to the biphasic course of COVID-19, therapeutic approaches that augment antiviral immunity may be more beneficial early in infection, whereas later interventions should focus on inflammation suppression. In this study, we show that AMP5A inhibits TLR 7/8 signaling in myeloid cells, resulting in a decrease in inflammatory mediators associated with hyperinflammation and autoimmunity. Furthermore, data demonstrating that AMP5A activates immunomodulatory transcription factors found to be protective in lung disease is provided. These findings suggest that the modes and mechanisms of action of AMP5A are well suited to treat conditions involving dysregulated TLR 7/8 activation.

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

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-ß, 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-1ß 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-1ß, 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-1ß 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.

The novel immunomodulatory biologic LMWF5A for pharmacological attenuation of the “cytokine storm” in COVID-19 patients: a hypothesis

A common complication of viral pulmonary infections, such as in the ongoing COVID-19 pandemic, is a phenomenon described as a “cytokine storm”. While poorly defined, this hyperinflammatory response results in diffuse alveolar damage. The low molecular weight fraction of commercial human serum albumin (LMWF5A), a novel biologic in development for osteoarthritis, demonstrates beneficial in vitro immunomodulatory effects complimentary to addressing inflammation, thus, we hypothesize that LMWF5A could improve the clinical outcomes of COVID-19 by attenuating hyperinflammation and the potential development of a cytokine storm. A variety of human in vitro immune models indicate that LMWF5A reduces the production of pro-inflammatory cytokines implicated in cytokine storm associated with COVID-19. Furthermore, evidence suggests LMWF5A also promotes the production of mediators required for resolving inflammation and enhances the barrier function of endothelial cultures. A randomized controlled trial, to evaluate the safety and efficacy of nebulized LMWF5A in adults with Acute Respiratory Distress Syndrome (ARDS) secondary to COVID-19 infection, was developed and is currently under review by the Food and Drug Administration. If successful, this therapy may attenuate the cytokine storm observed in these patients and potentially reduce mortality, increase ventilation free days, improve oxygenation parameters and consequently lessen the burden on patients and the intensive care unit. In conclusion, in vitro findings suggest that the immunomodulatory effects of LMWF5A make it a viable candidate for treating cytokine storm and restoring homeostasis to the immune response in COVID-19.