LncRNA AFAP1-AS1 Conveyed by Serum Exosomes from Patients with Septic Shock Promotes M1 Macrophage Polarization by Activating MAPK Signaling

Background: Septic shock, the most severe form of sepsis syndrome, is life-threatening and requires immediate medical attention. However, due to its poorly understood mechanism, there is no effective treatment for septic shock. Serum exosomes are impor-tant mediators of systemic inflammation due to septic shock, and we sought to uncover key signaling molecules in mediation. Methods: Serum exosomes from patients with septic shock (SS-exo) and healthy controls (Normal-exo) were isolated and char-acterized by western blot, nanoparticle tracking assay (NTA) and transmission electron microscopy (TEM). The long noncoding RNA (lncRNA) actin filament associated protein 1-antisense RNA 1 (AFAP1-AS1) expression levels in exosomes and THP-1 (the human leukemic cell line) cells co-incubated with the exosomes were measured by real-time quantitative reverse transcription PCR (polymerase chain reaction) (qRT-PCR). THP-1 cells were divided into lipopolysaccharide (LPS, 100 ng/mL) group, SS-exo group, SS-exo-shRNA group, and SS-exo-shRNA-AFAP1-AS1 group. The enzyme-linked immunosorbent assay (ELISA) was employed to examine the inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) expression in each group. Flow cytometry was applied to examine cell surface antigens CD86 (cluster of differentiation 86) and CD206 expres-sion in each group. In addition, qRT-PCR was conducted to determine IL-6, inducible nitric oxide synthase (iNOS), Arginase I (Arg1), and interleukin-10 (IL-10) expression levels in each group, and western blotting was employed to examine p-ERK1 (p-extracellular regulated protein kinases 1), ERK1/2, p38 MAPK (mitogen-activated protein kinase), and p-p38 MAPK protein expression levels in each group. Results: We successfully isolated and characterized SS-exo. The AFAP1-AS1 content was significantly increased in SS-exo. AFAP1-AS1 could be transmitted to THP-1 cells and stimulate THP-1 cell polarization to pro-inflammatory macrophages (M1 macrophages), thereby promoting the production of inflammatory factors IL-6 and TNF-alpha. Western blot revealed that AFAP1-AS1 significantly activated the MAPK signaling pathway. Conclusions: AFAP1-AS1, carried by SS-exo, excites THP-1 cell polarization in M1 macrophages and promotes an inflammatory response by activating the MAPK signaling pathway.

Automated summary

In this study, it was found that the long noncoding RNA AFAP1-AS1 in serum exosomes of patients with septic shock plays an important regulatory role in activating the MAPK signaling pathway, thereby promoting the inflammatory response of leukocytes.