It is unclear whether membrane vitamin D receptor (mVDR) exists on the macrophage membrane or whether mVDR is associated with lipopolysaccharide (LPS) tolerance. Herein, we report that interfering with caveolae and caveolae-dependent lipid rafts inhibited the formation of LPS tolerance. VDR was detected as co-localized with membrane molecular markers. VDR was detected on the cell membrane and its level was higher in LPS-tolerant cells than that in only LPS treatment cells. Anti-VDR antibodies could abolish the effect of artesunate (AS) to reverse LPS tolerance, and the wild-type peptides (H397 and H305) of VDR, but not the mutant peptide (H397D and H305A), led to the loss of AS’s effect. AS decreased the mVDR level in LPS-tolerant cells. In vivo, AS significantly reduced VDR level in the lung tissue of LPS-tolerant mice. In summary, mVDR exists on the cell membrane of macrophages and is closely associated with the formation of LPS tolerance and the effects of AS.

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The K_D value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

Kukoamine B (KB) was found to inhibit the over-production of pro-inflammatory cytokines induced by immunostimulatory CpG DNA, thus being considered as a valuable candidate for the development of anti-sepsis drugs. With the goal of elucidating the mechanism of inhibition of CpG DNA by KB, dual polarization interferometry and isothermal titration calorimetry were used to measure the kinetic and thermodynamic parameters of KB–CpG DNA interaction, and molecular docking was employed to predict the binding mode. The significant binding of KB to CpG DNA led to a conformational change of the CpG DNA layer on the biosensor chip surface and produced a dissociation equilibrium constant (K_D) of 4.82 × 10⁻⁷ M. The thermodynamic profile elucidated two-site binding, a strong binding of the first site and weaker binding of the second one, which was driven by enthalpy and entropy. Further, docking results suggested that KB formed a close contact with the core sequence of CpG DNA. Taken together with these results, we gained insight into the inhibition mechanism of KB against CpG DNA through direct binding measurement and in silico simulation. KB could bind the primary structure of CpG DNA, thereby interfering with the interaction of CpG DNA with Toll-like receptor 9 (TLR9) and the immunestimulatory effect.

Background and Purpose: Immunosuppression is the predominant cause of mortality for sepsis due to failure to eradicate invading pathogens. Unfortunately, no effective and specific drugs capable of reversing immunosuppression are available for clinical use. Increasing evidence implicates vitamin D receptor (VDR) involved in sepsis-induced immunosuppression. Herein, artesunate (AS) was discovered to reverse sepsis-induced immunosuppression and its molecular mechanism is investigated. Experimental Approach: Effect of artesunate on sepsis-induced immunosuppression was investigated in mice and in vitro . VDR was predicted to be an interacted candidate of AS by bioinformatics predict, then identified using PCR and immunoblotting. VDR, ATG16L1 and NF-κB p65 were modified to investigate the alteration of AS’s effect on pro-inflammatory cytokines release, bacteria clearance and autophagy activities in sepsis-induced immunosuppression. Key Results: AS significantly reduced the mortality of cecal ligation and puncture (CLP)-induced sepsis immunosuppression mice challenged with Pseudomonas Aeruginosa , and enhanced proinflammatory cytokines release and bacterial clearance to reverse sepsis-induced immunosuppression in vivo and in vitro . Mechanically, AS interacted with VDR thereby inhibited the nuclear translocation of VDR, then influencing ATG16L1 transcription and subsequent autophagy activity. In addition, AS inhibited physical interaction between VDR and NF-κB p65 in LPS tolerance macrophages, then promoted nuclear translocation of NF-κB p65, which activated the transcription of NF-κB p65 target genes such as pro-inflammatory cytokines. Conclusion and Implications: Our findings provide an evidence that AS interacted with VDR to reverse sepsis-induced immunosuppression in an autophagy and NF-κB dependent way, highlighting a novel approach for sepsis treatment and drug repurposing of AS in the future.