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

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.

CLINICALLY DISTINCT COVID-19 CASES SHARE STRIKINGLY SIMILAR IMMUNE RESPONSE PROGRESSION: A FOLLOW-UP ANALYSIS (preprint)

Inflammatory responses to the novel coronavirus SARS-CoV-2, which causes COVID-19, range from asymptomatic to severe. Here we present a follow-up analysis of a longitudinal study characterizing COVID-19 immune responses from a father and son with distinctly different clinical courses. The father required a lengthy hospital stay for severe symptoms, whereas his son had mild symptoms and no fever yet tested positive for SARS-CoV-2 for 29 days. Father and son, as well as another unrelated COVID-19 patient, displayed a robust increase of SERPING1, the transcript encoding C1 esterase inhibitor (C1-INH). We further bolstered this finding by incorporating a serum proteomics dataset and found that serum C1-INH was consistently increased in COVID-19 patients. C1-INH is a central regulator of the contact and complement systems, potentially linking COVID-19 to complement hyperactivation, fibrin clot formation, and immune depression. Furthermore, despite distinct clinical cases, significant parallels were observed in transcripts involved interferon and B cell signaling. As symptoms were resolving, widespread decreases were seen in immune-related transcripts to levels below those of healthy controls. Our study provides insight into the immune responses of likely millions of people with extremely mild symptoms who may not be aware of their infection with SARS-CoV-2 and implies a potential for long-lasting consequences that could contribute to reinfection risk.