Comprehensive transcriptomic analyses identify the immunosuppressive effects of LLDT-8 in ART-treated SIV-infected rhesus macaques.

BACKGROUND: Chronic immune activation plays a significant role in the pathogenesis and disease progression of human immunodeficiency virus (HIV), and the existing interventions to address this issue are limited. In a phase II clinical trial, (5R)-5-hydroxytriptolide (LLDT-8) demonstrated promising potential in enhancing CD4+ T cell recovery. However, the therapeutical effects of LLDT-8 remained to be systemic explored. METHODS: To assess the treatment effects of LLDT-8, we conducted flow cytometry and RNA-seq analyses on eight Chinese rhesus monkeys infected with simian immunodeficiency virus (SIV). Additionally, we performed comprehensive transcriptomic analyses, including cross-sectional and longitudinal differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and deconvolution analysis using peripheral blood mononuclear cell (PBMC) samples from 14-time points. These findings were further validated with RNA-seq analysis on patients who received LLDT-8 treatment, along with in vitro cellular experiments using human PBMCs. RESULTS: Flow cytometry analysis revealed that LLDT-8 treatment significantly reduced the percentage of HLA-DR+CD38+CD8+ T cells in SIV-infected rhesus monkeys (P < 0.001). The cross-sectional and longitudinal analysis identified 2531 and 1809 DEGs, respectively. GSEA analysis indicated that LLDT-8 treatment led to significant downregulation of proliferation-related pathways, such as E2F targets, G2M checkpoint, and mitotic spindle pathways. WGCNA analysis identified two modules and 202 hub genes associated with CD8 activation levels. Deconvolution analysis showed a significant decrease in the proportion of CD8+ T cells and activated CD4+ T cells during LLDT-8 treatment. Gene ontology results demonstrated that the common DEGs between LLDT-8-treated patients and rhesus monkeys were primarily enriched in cell activation and cell cycle progression. Furthermore, in vitro cellular experiments validated the consistent impact of LLDT-8 in inhibiting proliferation, activation (HLA-DR and CD38 expression), exhaustion (PD-1 expression), and IFN-γ production in human CD4+ and CD8+ T cells. CONCLUSION: LLDT-8 exhibited notable efficacy in alleviating immune activation in both an in vivo animal model and in vitro human cell experiments. These findings suggest that LLDT-8 may hold potential as a drug for managing systemic immune activation associated with SIV/HIV infection, warranting further prospective clinical exploration.

LLDT-8 ameliorates experimental autoimmune encephalomyelitis by mediating macrophage functions in the priming stage.

Multiple sclerosis (MS) is an inflammatory demyelinating disease in the central nervous system caused by T cell activation mediated by peripheral macrophages, resulting in severe neurological deficits and disability. Due to the currently limited and expensive treatments for MS, we here introduce an economic Chinese medicine extract, (5R)-5-Hydroxytriptolide (LLDT-8), which shows low toxicity and high immunosuppressive activity. We used the widely accepted mouse model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the immunosuppressive effect of LLDT-8 in vivo. Through the RNA-sequence analysis of peripheral macrophages in EAE mice, we discovered that LLDT-8 alleviates the symptoms of EAE by inhibiting the proinflammatory effect of macrophages, thereby blocking the activation and proliferation of T cells. In all, we found that LLDT-8 could be a potential treatment for MS.

Therapeutic effect of (5R)-5-hydroxytriptolide (LLDT-8) in SIV infected rhesus monkeys.

BACKGROUNDS: Human immunodeficiency virus (HIV) infections induce robust, generalized inflammatory responses and lead to pathological systemic immune activation. This abnormal immune status persists despite successful antiretroviral therapy (ART). Immune modulating strategies in conjunction with ART were tried to reduce abnormal immune activation. Previously, we demonstrated that Tripterygium Wilfordii Hook F has been shown immunosuppressive activity in HIV patients. (5R)-5-hydroxytriptolide (LLDT-8), a new analog of triptolide, and the most active ingredient of Tripterygium Wilfordii Hook F, has been shown to have lower cytotoxicity. However, the role of LLDT-8 in HIV or simian immunodeficiency virus (SIV) needs to be explored. METHODS: Six male adult Chinese rhesus monkeys were enrolled in our study. All of them were healthy and negative for SIV, and chronically SIVmac239 infected macaques were treated with LLDT-8 combined with ART (n = 4) or ART only (n = 2) after 14 weeks of infection. ART was determined at week 33, and LLDT-8 was continued until week 48. T cell immune activation and inflammation were compared during the period, and viral rebound time and reservoir were supervised after stopping ART. RESULTS: The RNA level of the two groups continued to decline after initiating ART, RNA of 4 rhesus monkeys declined to the lower limit of detection at week 20. LLDT-8 administration combined with ART did not affect T cell activation and plasma levels of IL-6 and CRP. The viral load of all the macaques in both groups was rebounded 2 weeks after ART discontinuation. Furthermore, no significant decrease of SIV DNA was observed in the LLDT-8 treatment group. CONCLUSIONS: LLDT-8 administration during chronic SIV infection had no effect on T cell activation and plasma levels; Furthermore, LLDT-8 may not contribute to suppression of viral rebound and reservoir. These results suggest that LLDT-8 is unlikely to reduce immune activation and viral persistence without additional interventions.

Tripterygium wilfordii derivative LLDT-8 targets CD2 in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory synovitis systemic disease, can lead to joint deformities, loss of function and even death. The pathogenesis of RA may be related to genetics, infection and/or sex hormones; however, detailed accounts of the molecular mechanisms underlying its pathogenesis are lacking. In the present study, the synovial tissues of patients with RA and healthy individuals were analyzed to identify the pathogenic signaling pathways and key candidate genes involved in RA. Gene Ontology (GO), pathway enrichment and protein-protein interaction analysis were further used to identify the differentially expressed genes (DEGs) and their potential roles in RA. Molecular docking was used to screen the potential candidate drugs for management of RA. Small interfering RNA was used for knockdown of the CD2 protein. A Cell Counting Kit-8 assay was used to detect the proliferation of cells. Changes in the levels of inflammatory cytokines were detected using ELISA. A total of 279 DEGs were identified in RA; amongst these genes, 166 and 113 were upregulated and downregulated, respectively. GO analysis revealed that the upregulated DEGs were primarily enriched in the activation of the immune and adaptive immune responses, as well as the inflammatory response. The T-cell surface antigen CD2 (CD2) was identified as the most important hub gene by selecting the most important module from the protein-protein interaction network. Knockout of CD2 reduced the damaging effects of TNF-α on synovial cells. Through in situ screening using computer-aided drug design, the triptolide derivative (5R)-5-hydroxytriptolide (LLDT-8) was determined to have the highest docking score based on the CD2 protein structure. Cell experiments showed that LLDT-8 could inhibit the expression of CD2. Cell proliferation and inflammatory cytokine assays confirmed that CD2 was the direct target of LLDT-8. Together, the results of the present study determined factors involved in the pathogenesis of RA and the important role of CD2 in this process by analyzing the DEGs in the RA process. LLDT-8 inhibited CD2 and may thus be used to treat RA. These candidate genes and signaling pathways may serve as potential targets for the clinical treatment of RA.

Triptolide analog LLDT-8 ameliorates psoriasis-like dermatitis in BALB/c mice via suppressing the IL-36α signaling pathway.

Triptolide has shown a good immunosuppressive effect on autoimmune diseases. However, the toxicity limited its widely clinical practice. In this study, we investigated the effects and underlying mechanisms of (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative, on a murine psoriasis-like dermatitis model and related cell lines. Here, we showed that LLDT-8 significantly attenuated symptoms of psoriasis-like dermatitis induced by imiquimod (IMQ, a TLR7 agonist) by reducing the psoriasis area and severity index (PASI) score and inflammatory parameters. The action of LLDT-8 was involved in down-regulated interleukin (IL)-36α expression and blocked IL-36α pathway by LC-MS-based label-free quantitative (LFQ) proteomic approach and further experiments. Meanwhile, we observed that LLDT-8 significantly inhibited the expression of IL-36α in R837-treated bone marrow-derived dendritic cells (BMDCs). In conclusion, LLDT-8 notably alleviated IMQ-induced psoriasis-like skin inflammation via suppressing the IL-36α signaling pathway, suggesting LLDT-8 might be a potential drug for the treatment of psoriasis.

(5R)-5-hydroxytriptolide (LLDT-8) prevents collagen-induced arthritis through OPG/RANK/RANKL signaling in a rat model of rheumatoid arthritis.

(5R)-5-hydroxytriptolide (LLDT-8) extracts from Tripterygium have anti-inflammatory, antineoplastic and immunity adjustment functions. The present study used a collagen-induced arthritis (CIA) model to evaluate whether LLDT-8 prevents collagen-induced arthritis, and investigated the signaling underlying this. Male Sprague-Dawley rats were induced to generate CIA, mimicking rheumatoid arthritis (RA). The presence of arthritis was determined using RA progression scores. The inflammatory cytokines interleukin (IL)-1ß, IL-6 and nuclear factor-κB were detected using enzyme-linked immunosorbent assay kits. Induced nitric oxide synthase (iNOS) and matrix metalloprotease (MMP)-13 protein expression were measured using western blot analysis. Lastly, reverse transcription-quantitative polymerase chain reaction was used to evaluate osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) gene expression. LLDT-8 improved RA progression scores and reduced the incidence and severity of CIA. Furthermore, LLDT-8 administration inhibited collagen-induced inflammation and iNOS protein expression in arthritic rats. The current data indicated that MMP-13 production was suppressed and OPG/RANKL expression was increased by LLDT-8 treatment in the arthritic rat. The present results suggest that LLDT-8 attenuates CIA through OPG/RANK/RANK ligand signaling in a rat model of RA.

LLDT-8 protects against cerebral ischemia/reperfusion injury by suppressing post-stroke inflammation.

AIM: (5R)-5-Hydroxytriptolide (LLDT-8), an analogue of triptolide, displays lower toxicity compared to triptolide and has comparable immunosuppressive effects. We investigated the anti-inflammatory and neuroprotective effects of LLDT-8 on cerebral ischemia/reperfusion injury. METHODS: Nitric oxide production from microglia was assessed by measuring the nitrite concentration in the culture medium with Griess reagent. Microglial cells and ischemic brain tissues were examined for the expression of proinflammatory mediators by qPCR and western blot. Infarct volumes were assessed with TTC histology. The TLR4/NF-κB signaling pathway was analyzed with western blot and immunocytochemistry. RESULTS: LLDT-8 significantly reduced infarct sizes and expression of pro-inflammatory cytokines in the ischemic cortex. LLDT-8 inhibited NO release and expression of TNF-α, IL-1ß and iNOS in BV-2 microglia and primary microglia treated with LPS. In addition, LLDT-8 suppressed expression of TLR4, degradation of IκBα and nuclear translocation of NF-κB. CONCLUSION: LLDT-8 exerted anti-inflammatory effects and protected against acute cerebral ischemia/reperfusion injury possibly by acting through the IκB/NF-κB cascade to suppress microglia-mediated neuroinflammation.