ENDOTARGET publication: β Boswellic Acid Blocks Articular Innate Immune Responses: An In Silico and In Vitro Approach to Traditional Medicine

Eloi Franco-Trepat, Ana Alonso-Pérez, María Guillán-Fresco, Miriam López-Fagúndez, Andrés Pazos-Pérez, Antía Crespo-Golmar, Susana Belén Bravo, Verónica López-López, Alberto Jorge-Mora, José P. Cerón-Carrasco, Ana Lois Iglesias and Rodolfo Gómez  

2023, Antioxidants

 

Background and Objectives: 

β Boswellic Acid (BBA) is a component of the Boswellia serrata extract (BSE) mix, which is commonly taken as an infusion or food complement in traditional ayurvedic medicine to treat inflammatory diseases such as asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis (OA). Interestingly, the BSE element BBA has recently been described to inhibit NFκB, a downstream signaling pathway of TLR4/IL1R. TLR4 and IL1R are two innate immune response receptors associated with the development of multiple rheumatic diseases. The IL1R has been used as a therapeutic target for diseases such as OA, rheumatoid arthritis or gout. However, there is no clinically available BBA-based TLR4 inhibitor to treat rheumatic inflammatory diseases. In the present study, the BBA molecule from the BSE mix was studied to elucidate its potential therapeutic effects on OA joint cells.

 

Results: 

Proteome Profile of TLR4/IL1R-Stimulated Primary Human OA Chondrocytes 

The first approach was to analyse the primary human OA chondrocytes secretome and proteome changes linked to TLR4 and IL1R activation to validate novel and already described inhibitory targets. Key data was obtained on the TLR4/IL1R downstream signaling pathways. Specifically, MAPK p38/NFκB, TNF, IFNαβ, ROS, and NLRP3 downstream pathways were found enriched. Similar results were obtained in mice ATDC5 chondrocytes proteome 

Predicting the Binding Affinity of BBA to Potential Proteome Profile Targets 

Through computational docking experiments the potential inhibition of these factors by BBA was investigated. A great binding affinity of BBA was predicted to the human TLR4 complex and NFκB. Furthermore, this binding affinity of BBA to TLR4 was found equivalent to known antagonists such as CLI-095. Interestingly, the docking analysis predicted that BBA could not bind to any domain of IL1R, NLRP3, or TLR3 complex. Although docking score results found the binding of BBA to TLR4 the most energetically and structurally stable, the BBA effective permeability coefficient does not discard BBA from crossing the cellular membrane and binding to NFκB. Nonetheless, molecular dynamics simulations presented additional evidence of BBA-TLR4 binding stability, suggesting that specific TLR4 inhibition may be the main mechanism of action. 

BBA Effects on Cell Viability and ROS Secretion in Chondrocytes 

In vitro experiments were conducted to validate the in silico predictions and to explore BBA’s potential modulation of the TLR4/IL1R signaling pathways, particularly focusing on reactive oxygen species (ROS) generation, which was found enriched in both human and mouse proteome profiles. Therefore, the study focused on NO generation, which is a common factor in both TLR4 and IL1R activation, with links to OA development and increased ROS production.  

At first, call viability and NO2-accumulation in primary hOC and mice ATDC5 chondrocytes were analysed. Upon stimulation with LPS and IL1β, cell viability remained unchanged, but NO2– accumulation increased. BBA at lower concentrations reduced NO2– levels in a dose-dependent manner without affecting cell viability. Next, the NOS2 gene expression was analysed. Low concentrations of BBA decreased NOS2 mRNA levels in a dose-dependent manner, consistent with reduced NO2– levels. Interestingly, gene expression of TLR4 and IL1R showed an increase in mRNA levels after LPS/IL1β stimulation but no modulation after BBA co-treatment, indicating that BBA effects were not bound to the modulation of TLR4 pr IL1R mRNA levels. Taken together, these results demonstrate an inhibitory effect of BBA in the NOS2-axis. 

BBA Effects on TLR4/IL1R-Mediated IIRs in Primary Human OA Chondrocytes, Human OA Chondrocytes and Human OA Synoviocytes 

The mRNA expression of common specific elements in the MAPK p38, and NFκB pathway in hOC was evaluated. These elements showed increased mRNA levels after TLR4/IL1R activation and decreased ones after cotreatment with BBA. Interestingly, gene expression of TLR4 and IL1R showed an increase in mRNA levels after LPS/IL1β stimulation but no modulation after BBA co-treatment. To validate this data, the intracellular and secreted levels of IL6 in these cells were evaluated. The TLR4/IL1R activation increased secreted and cellular protein concentration of IL6 while BBA cotreatment inhibited it. Consistent with a decreased activation of MAPK p38/NFκB pathway by BBA, a reduction in the total phosphoproteome after BBA treatment was observed in TLR4/IL1R-activated hOC. On the transcriptomic level, inflammatory factors and catabolic factors mRNA levels were increased after LPS and IL1β stimulation of primary hOC, whereas anabolic factors were decreased. Gene expression of inflammatory and catabolic factors decreased in a dose-response manner after co-treatment with BBA, and anabolic factors increased and reverted to basal control levels.  

Compared to hOC, hOB presented a similar transcriptomic profile for ROS/NOS2, MAPK p38/NFκB pathway and TLR4/IL1R downstream signaling factors. The 48-h stimulation with LPS and IL1β increased NOS2, TLR4, IL1R, MAPK p38, and IL6 mRNA levels, as well as IL6 protein levels and the phosphoproteome status. Overall, the intensity of TLR4/IL1R-activation on hOB osteoblasts mRNA and protein levels were lower compared to hOC chondrocytes, but BBA co-treatment effects were the same. 

The same experiments were done with hOS. Overall, the intensity of TLR4/IL1R-activation on human synoviocytes mRNA and protein levels was higher compared to hOC chondrocytes and hOB osteoblasts, but BBA co-treatment effects were the same. 

BBA Effects in Joint Cells on the NLRP3 Inflammasome Pathway 

In TLR4/IL1R-activated hOC, gene expression of NLRP3, CASP1, and IL1β was highly increased. In contrast, treatment with BBA reduced the induced mRNA levels. Confirming these results, stimulation with LPS or IL1β increased CASP1 activity and cellular and secreted IL1β protein. In line with previous results, treatment with BBA reduced CASP1 activity and cellular and secreted IL1β levels to basal levels. This effect could also be replicated in hOB and hOS.

 

Conclusion:

In conclusion, this study shows evidence that BBA selectively binds to the TLR4 complex and inhibits in primary human joint cells the TLR4/lL1R-mediated innate immune responses associated with the activation of ROS synthesis, MAPK p38/NFκB, IFNαβ, TNF, and NLRP3 signaling pathways. Furthermore, it was determined in stimulated and non-stimulated articular cells that TLR4 inhibition by BBA or other known inhibitors is associated with NLRP3 downregulation. Moreover, it was revealed that this downregulation was enough to recapitulate the effects of BBA or TLR4 inhibition on TLR4/lL1R-mediated IIR. Altogether, the provided data supports the suggestion that BBA acts as a strong TLR4 inhibitor that may help to control OA-associated IIR. 

 

Read the full publication: β Boswellic Acid Blocks Articular Innate Immune Responses: An In Silico and In Vitro Approach to Traditional Medicine 

Glossary:

• β Boswellic Acid (BBA): a bioactive compound derived from the resin of Boswellia trees, known for its anti-inflammatory and anti-arthritic properties. 
• Boswellia serrata extract (BSE): a herbal supplement derived from the resin of the Boswellia serrata tree. 
• Caspase1 (CASP1): an enzyme that plays a crucial role in the inflammatory response by mediating the maturation and release of pro-inflammatory cytokines. 
• Chondrocyte: cartilage cells that synthesise and maintain the cartilaginous matrix in cartilage tissue and are thus crucial for its structure and function. 
• CLI-095: a small-molecule inhibitor that specifically targets TLR4, blocking its signaling pathway and thereby reducing inflammation. 
• Human OA chondrocytes (hOC): cartilage cells derived from patients with osteoarthritis. 
• Human OA synoviocytes (hOS): synoviocytes derived from patients with osteoarthritis. 
• Human osteoblasts (hOB): bone-forming cells derived from patients with osteoarthritis. 
• Innate immune response (IIR): refers to the human’s initial defense mechanism against pathogens, involving a rapid, non-specific response by various immune cells and molecules to detect and eliminate invaders. 
• Interferon-α/β (IFN-α/β): types of cytokines that play a critical role in the antiviral immune response by inhibiting viral replication and modulating the activity of immune cells. 
• Interleukin-1 beta (IL-1β): pro-inflammatory cytokine protein produced by activated immune cells (macrophages) and involved in various cellular activities, including inflammation and immune responses. 
• Interleukin-1 receptor (IL1R): a cell surface receptor that binds to IL-1 cytokines, playing a crucial role in mediating inflammatory and immune responses. 
• Interleukin-6 (IL6): a cytokine involved in regulating immune responses, inflammation, and hematopoiesis, playing a key role in the body’s defense mechanisms and inflammatory diseases. 
• p38 mitogen-activated protein kinases (MAPK/p38): enzyme involved in cellular signaling pathways that regulate inflammation, stress responses, and cell differentiation in response to various external stimuli. 
• Messenger RNA (mRNA): a molecule that carries genetic information from DNA to the ribosome, where it serves as a template for protein synthesis. 
• Mice ATDC5 chondrocytes: a cell line derived from mouse teratocarcinoma, commonly used as a model to study cartilage development and chondrocyte differentiation in vitro. 
• Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NFκB): a transcription factor that plays a key role in regulating immune responses, inflammation, and cell survival by controlling the expression of genes involved in these processes. 
• NLR family pyrin domain containing 3 (NLRP3): cytosolic receptor that is crucial for initiating the assembly of the inflammasome and triggering inflammation in response to various stimuli. 
• Nitric oxide synthase (NOS2): an enzyme responsible for catalyzing the production of nitric oxide (NO) from arginine and oxygen. 
• Osteoarthritis (OA): degenerative joint disease characterised by the breakdown of cartilage, leading to pain, swelling, stiffness, and reduced mobility in affected joints. 
• Phosphoproteome: complete set of phosphorylated proteins within a cell, tissue, or organism, which is studied to understand protein function and signaling pathways regulated by phosphorylation. 
• Proteome: complete set of proteins expressed by a cell, tissue, or organism at a given time, reflecting its functional state and biological processes. 
• Reactive oxygen species (ROS): highly reactive molecules containing oxygen that are generated as natural byproducts of cellular metabolism, playing roles in signaling and oxidative stress. 
• Secretome: complete set of proteins and molecules secreted by a cell, tissue, or organism, playing crucial roles in communication, signaling, and interactions with the extracellular environment. 
• Synoviocytes: specialised cells that line the inner layer of the synovial membrane in joints, responsible for producing synovial fluid and maintaining the health of joint tissues. 
• Toll-like receptor 4 (TRL4): is a protein on the surface of immune cells that recognises specific bacterial molecules and triggers an immune response to defend against infections. 
• Tumor necrosis factor (TNF): a cytokine involved in systemic inflammation, immune regulation, and apoptosis, playing a significant role in various inflammatory and autoimmune diseases.