Abstract

   Osteoarthritis (OA) is hallmarked as a silent progressive rheumatic
   disease of the whole joint. The accumulation of inflammatory and
   catabolic factors such as IL6, TNFα, and COX2 drives the OA
   pathophysiology into cartilage degradation, synovia inflammation, and
   bone destruction. There is no clinical available OA treatment. Although
   traditional ayurvedic medicine has been using Boswellia serrata
   extracts (BSE) as an antirheumatic treatment for a millennium, none of
   the BSE components have been clinically approved. Recently, β boswellic
   acid (BBA) has been shown to reduce in vivo OA-cartilage loss through
   an unknown mechanism. We used computational pharmacology, proteomics,
   transcriptomics, and metabolomics to present solid evidence of BBA
   therapeutic properties in mouse and primary human OA joint cells.
   Specifically, BBA binds to the innate immune receptor Toll-like
   Receptor 4 (TLR4) complex and inhibits both TLR4 and Interleukin 1
   Receptor (IL1R) signaling in OA chondrocytes, osteoblasts, and
   synoviocytes. Moreover, BBA inhibition of TLR4/IL1R downregulated
   reactive oxygen species (ROS) synthesis and MAPK p38/NFκB, NLRP3,
   IFNαβ, TNF, and ECM-related pathways. Altogether, we present a solid
   bulk of evidence that BBA blocks OA innate immune responses and could
   be transferred into the clinic as an alimentary supplement or as a
   therapeutic tool after clinical trial evaluations.

   Keywords: osteoarthritis, chondrocytes, synoviocytes, ROS, TLR4, IL1,
   NLRP3

1. Introduction

   The worldwide number of patients suffering from rheumatic diseases such
   as osteoarthritis (OA) equals the whole United States of America (USA)
   population. That is more than 300 million people that will suffer their
   whole life from daily chronic pain and disability with no option for an
   efficient treatment [[46]1,[47]2,[48]3]. Currently, OA is defined as a
   complex heterogenic disease [[49]4,[50]5,[51]6] with no clinical
   [[52]7,[53]8,[54]9] or molecular [[55]10,[56]11,[57]12] consensus on
   the disease pathophysiology. It is agreed to be a silent progressive
   degradative disease that hinders the early diagnosis of articular
   cartilage degradation, synovial inflammation [[58]13,[59]14], and/or
   bone alteration [[60]15,[61]16]. Then, biomechanical, metabolic, and
   aging-related alterations accumulate to the point that the whole joint
   space narrows due to cartilage destruction [[62]17,[63]18,[64]19].
   Chronic articular destruction activates inflammatory, catabolic, and
   pain pathways that lead to acute pain that might bring permanent
   disability [[65]17,[66]18,[67]20]. Great efforts have been committed to
   different drug discovery approaches; pharmacomodulation [[68]21], drug
   repurposing [[69]22,[70]23], and the re-discovery of natural products
   from traditional medicine, namely, the first Chinese medicine book
   Hunagti Neiching [[71]24] and the Indian Ayurvedic medicine [[72]25].

   Indian traditional medicine has preserved a rich repertoire of
   antiarthritic remedies including Amalaki (Embelia officinale), Maricha
   (Piper nigrum), Turmeric (Curcuma longa) [[73]26], and Boswellia
   serrata extract (BSE) mix [[74]27]. Furthermore, several studies have
   shown that the Indian traditional remedy BSE mix inactivates multiple
   immune-related pathways [[75]28]. Nonetheless, the lack of a normalized
   composition for the BSE mix [[76]29,[77]30] has caused several
   inconsistencies in the potential therapeutic effects on rheumatic
   diseases such as OA and Rheumatoid Arthritis (RA). This paradigm might
   change via the standardized BSE mix initiative Casperome [[78]31].
   Although most studies suggest that the BSE mix contains nuclear factor
   kappa-light-chain-enhancer of activated B cells (NFκB) inhibitors
   [[79]32,[80]33], other studies suggest that NFκB inhibition might
   indirectly be via 5-lipoxygenase [[81]27,[82]33] or reactive species of
   oxygen (ROS)-related processes [[83]34,[84]35]. Research on individual
   BSE mix components such as 11-Keto-β-Boswellic Acid (KBA) and
   Acetyl-11-Keto-β-Boswellic Acid (AKBA) is very limited; nevertheless,
   anti-inflammatory and anticatabolic effects were found consistently
   across the different in vitro, in vivo, and food supplement studies
   [[85]33,[86]36,[87]37]. Interestingly, onlu 3% of the BSE mix studies
   have focused on the BSE mix component β Boswellic Acid (BBA), also
   known as beta-BA. Moreover, BBA was able to block cartilage loss and
   inflammatory factors in Toll-like Receptor 4 (TLR4)/Interleukin 1
   Receptor (IL1R)-activated synovial explant tissues [[88]27].
   Nonetheless, little is known about the specific role of BBA on TLR4 or
   IL1R receptors.

   The innate immune receptor TLR4 has not been fully explored as a
   therapeutic target in the clinical rheumatology field [[89]38],
   nonetheless, its signaling crosstalks with IL1R [[90]39,[91]40,[92]41]
   a well-explored therapeutic target for OA
   [[93]42,[94]43,[95]44,[96]45,[97]46,[98]47,[99]48,[100]49] and RA
   [[101]50,[102]51]. The degradation in the OA joint predominantly
   affects the cartilage and releases damage-associated molecular patterns
   (DAMPs) [[103]17,[104]52] that activate TLR4 signaling. In contrast,
   IL1R is activated by the accumulation of cytokine interleukin 1β
   (IL1β). The downstream signaling of both receptors meet in myeloid
   differentiation primary response 88 (MyD88), a molecule involved in the
   signal transduction and activation of NFκB [[105]53]. The NFκB pathway
   modulates several OA-associated innate immune factors such as nitric
   oxide synthase 2 (NOS2), cyclooxygenase-2 (COX2), interleukin 6 (IL6),
   and matrix metalloproteinase 3 (MMP3) or 13 (MMP13)
   [[106]54,[107]55,[108]56] that cause inflammation and tissue
   degradation. Remarkably, BBA has been shown to inhibit the NFκB pathway
   in osteoblasts [[109]57], macrophages [[110]32], and other cell types
   [[111]58,[112]59,[113]60]. Nonetheless, there is a lack of data on BBA
   inhibition of NFκB in chondrocytes and cartilage.

   In the present study, we selected the understudied BBA molecule from
   the BSE mix to finely elucidate the potential therapeutic effects on OA
   joint cells. Moreover, we made use of computational chemistry, a new
   current trend in drug discovery screening [[114]61,[115]62], to
   demonstrate the higher binding affinity [[116]63] between BBA and TLR4
   compared to other potential target candidates, as well as the ability
   of BBA to dock the TLR4 receptor in a passive and more advanced dynamic
   in silico model. Furthermore, we demonstrated that this antagonism of
   the TLR4 complex inhibited TLR4 and IL1R-associated innate immune
   responses in chondrocytes, osteoblasts, and synoviocytes isolated from
   OA patients. Moreover, we propose a mechanism of action to support the
   inhibitory effect of BBA on both receptors.

   All in all, the use of BBA as a nutraceutical in clinical practice
   might be useful in the management of degradative joint processes
   observed in OA patients.

2. Materials and Methods

2.1. Docking Analysis

   Molecular 3D structures of β boswellic acid (BBA), CLI-095 or TAK242,
   myristic acid, 2-OH myristic acid, 3-OH myristic acid, and lauric acid
   were obtained from the PubChem database [[117]64], whereas NFκB (1SVC),
   IκB kinase (IKK) (3RZF), NLR family pyrin domain containing 3 (NLRP3)
   (6NPY), Toll-like Receptor 3 (TLR3) (3CIY), TLR4 (3VQ1), and IL1R1
   (1ITB) biomolecular targets were obtained from protein data bank (PDB)
   [[118]65]. Remarkably, Xu et al. deposited the IKK (3RZF) crystal
   [[119]66] and resolved the active-site residues (Leucine 21 (Leu21),
   Glycine 22 (Gly22), Threonine 23 (Thr23), Valine 29 (Val29), Alanine 42
   (Ala42), Lysine 44 (Lys44), Methionine (Met96), Glutamine (Glu97),
   Tyrosine 98 (Tyr98), Cystine 99 (Cys99), Gly102, Asparagine 103
   (Asp103), Glu149, Asparagine 150 (Asn150), Isoleucine 165 (Ile165), and
   Aspartic Acid 166 (Asp166) in presence of the Cmpd1 inhibitor known as
   (4-{[4-(4-Chlorophenyl) pyrimidin-2-Yl] amino} phenyl)
   [4-(2-Hydroxyethyl) piperazin-1-Yl] methanone. Similarly, Mao et al.
   deposited the NLRP3 (6NPY) crystal [[120]67] that resolves the
   adenosine diphosphate (ADP) binding site and has successfully been used
   for virtual screening simulations [[121]68,[122]69]. All protein
   database (PDB) crystallographic structures were refined with the
   protein preparation wizard module implemented in the Schrödinger suite
   [[123]70]. A grid was generated at the ligand sites to attempt a
   molecular docking using the extra-precision (XP) scoring functions
   implemented in Glide [[124]71,[125]72]. Binding energies were computed
   at the molecular mechanics (MM)/generalized born surface area (GBSA)
   [[126]73,[127]74] following the described theory [[128]75]. Additional
   molecular dynamics (MD) calculations were performed, aiming to confirm
   the predicted stability of the best-ranked drug-target system.
   Specifically, MD trajectories were generated with the graphic
   processing unit (GPU)-accelerated version of Desmond that successfully
   allows them to mimic interactions in a drug design [[129]76]. The MD
   simulation is completed by producing trajectories of 100 ns. All
   calculations were conducted with Schrödinger (Schrödinger Release
   2021-3).

2.2. Cell Culture

   The Santiago Hospital Ethics Committee (CAEIG-2016/258) approved the
   informed consent, the signature for which is mandatory for all
   participant donors. Articular issue samples from healthy and OA
   patients were, respectively, obtained from dead and total knee
   replacement surgery donors [[130]77]. Primary joint cells
   (chondrocytes, osteoblasts, and synoviocytes), isolated from their
   respective articular tissues, murine chondrogenic ATDC5 (RIKEN Cell
   Bank, Tsukuba, Japan), human osteoblastogenic SaOs2, and human
   synovial-sarcoma SW982 (ATCC, Manassas, VA, USA), were initially
   cell-cultured in P100 plates with DMEN F12 cell medium supplemented
   with fetal bovine serum (FBS) at 5% (mouse cells) or 10% (human cells),
   2% Glutamine, and 2% penicillin-streptomycin, plus 0.2% sodium selenite
   and 0.1% transferrin (mouse cells) [[131]22,[132]23,[133]77,[134]78].

   Only early cell passes (P) comprised of P[0]–P[3] (primary cells) and
   P[5]–P[35] (non-primary cells) were used to keep the cellular phenotype
   unaltered. Cells were seeded in cell plates (P12 or P100) and after 4
   h, the medium was renewed with FBS deprived medium
   [[135]22,[136]23,[137]77,[138]78]. All cell culture reagents were
   purchased from Sigma-Aldrich (Sant Louis, MO, USA) except otherwise
   mentioned.

2.3. Cell Treatment

   The concentration of use for lipopolysaccharide E. coli 026:B6 (LPS)
   (100 ng/mL), a pathogen-associated molecular pattern (PAMPs), and IL1β
   (0.1 ng/m) (Tebu-Bio, France) was selected from the literature
   [[139]22] to be used, respectively, as a canonical agonist for TLR4 and
   IL1R complex. The specific TLR4 antagonist CLI-095/TAK-242 (1.00 µM)
   (InvivoGen, San Diego, CA, USA) was used as a TLR4 inhibitor
   [[140]22,[141]79]. We selected low concentrations of BBA (0.10–5.00 µM)
   (Merck, Darmstadt, Germany) to facilitate clinical transferability. All
   cell culture reagents were purchased from Sigma-Aldrich (Sant Louis,
   MO, USA) except otherwise mentioned. PubChem: IL1β CID 159483, CLI-095
   CID 9919285, BBA CID 168928.

2.4. Metabolic and Biochemical Assays

   The accumulation of nitrite (NO[2]^−) levels was measured by Griess
   assay (Merck, Darmstadt, Germany) [[142]22,[143]77]. Nitrite
   measurement serves as an indirect determination of the innate immune
   response factor and ROS element, the nitric oxide (NO). Cell viability
   was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
   bromide (MTT) assay (Merck, Darmstadt, Germany) [[144]22,[145]77].
   Treatment concentrations were adjusted upon MTT and Griess assay
   dose-response results. Caspase 1 (CASP1) enzymatic activity was
   determined by Ac-Valine-Alanine-Asparagine-pNA (pNA) assay (Abbkine,
   Wuhan, China), and phosphoproteome was determined by Green Malachite
   assay (Merck, Darmstadt, Germany).

2.5. Transcriptomic Silencing

   A specific short interference ribonucleic acid RNA (siRNA) was used to
   transiently silence the messenger RNA (mRNA) gene expression of NLRP3
   (siNLRP3) and TLR4 (siTLR4) [[146]80]. Briefly, cells were plated at a
   density of 50,000 cells/well (P24 plate), washed with Optimen and DMEN
   F12 (2% FBS) after 4h, and incubated for 1h with DMEN F12 (2% FBS).
   Then siRNA mix (15 nM) was administered for 24 h before pharmacological
   treatments such as LPS and IL1β. Both TLR4 and NLRP3 siRNA mix (15 nM)
   from TriFECTa DsiRNA kit (IDT, Coralville, IA, USA) contained 3
   different oligos (sequence data not available) in equal proportion.
   Silencing effectiveness and specificity were validated in each
   experiment (n = 6) through positive (Hypoxanthine Guanine
   Phosphoribosyltransferase S1; HPRT-S1) and negative (scramble)
   controls. We confirmed the mRNA levels reduction of HPRT (71–92%) by
   positive controls, as well as TLR4 and NLRP3 (61–81%) by siTLR4 and
   siNLRP3 treatments. Furthermore, we confirmed the inability of negative
   controls to knock down NLRP3, TLR4, or HPRT mRNA levels.

2.6. Transcriptomic Profiling

   The gene expression (mRNA) of multiple genes ([147]Supplementary data
   S1) was analyzed by comparative reverse transcription (RT)—quantitative
   polymerase chain reaction (qPCR) [[148]22,[149]77]. Briefly, the mRNA
   was isolated from cell cultures (2.5 × 10^5 cells/well) using the
   trizol-chloroform column-based protocol of EZNA Total RNA Kit I (Omega
   Bio-Tek, Norcross, GA, USA) [[150]77,[151]81]. The mRNA was purified
   using deoxyribonucleic acid (DNA) enzyme DNAsa I, normalized and
   quantified by Nanodrop (260 nm). The mRNA (500 µg) was retrotranscribed
   to copy DNA (cDNA) using the High-Capacity RNA-To-cDNA Kit (Thermo
   Fisher Scientific, Waltham, MA, USA) [[152]82]. The RT-qPCR
   determinations were performed under the minimum Information for
   publication of quantitative real-time PCR experiments (MIQE) guidelines
   [[153]83]. Briefly, the RT-qPCRs were performed using cDNA, iTaq
   Universal SYBR Green Supermix (BioRad, Hercules, CA, USA), and
   KiCqStart SYBR Green primers (Merck, Darmstadt, Germany). These include
   TLR4, IL1R1, Heatshock Protein 90 αα1 (HSP90AA1), Mitogen-activated
   protein kinase (MAPK) 14 (MAPK14) or p38, CASP1, Nucleotide-binding
   oligomerization domain, NLRP3, IL6, IL1β, lipocalin 2 (LCN2),
   cyclooxygenase 1 (COX1), COX2, intercellular adhesion molecule 1
   (ICAM1), vascular cell adhesion protein 1 (VCAM1), monocytes
   chemoattractant protein 1 (MCP1), and nicotinamide phosphoribosyl
   transferase (NAMPT), as well as catabolic factors such as
   metalloproteinase 1 (MMP1), -3 (MMP3), -9 (MMP9), -13 (MMP13), and A
   disintegrin and metalloproteinase with thrombospondin motifs 4
   (ADAMTS4), aggrecan (ACAN), collagen 1 α1 (COL1A1), and collagen 2 α1
   (COL2A1).

   Furthermore, we used dependent or endogen control (Low-ROX dye) and the
   independent reference control (hypoxanthine phosphoribosyl transferase
   1; HPRT1). The RT-qPCRs were performed under a FAST thermal profile (95
   °C for 20″; 95 °C for 1″ and 60 °C for 20″ during 45 cycles) in a
   QuantStudio3 (QS3) thermocycler (Thermo Fisher Scientific, Waltham, MA,
   USA) [[154]84,[155]85].

2.7. Proteome Profiling

   Protein levels were analyzed by enzyme-linked immunosorbent assay
   (ELISA) and matrix-assisted laser desorption/ionization (MALDI)—time of
   flight (TOF). Briefly, proteins were isolated from cell cultures using
   RIPA 1X buffer (Merck, Darmstadt, Germany) with HALT protease and
   phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) and
   quantified using the bovine serum albumin (BSA) Pierce Bradford assay
   (ThermoFisher, USA).

   The cellular and secreted (supernatant) levels of IL6 and IL1β proteins
   were determined in joint cells by mouse and human ELISA antibodies
   (Abbkine, Wuhan, China) and quantified (450 nm and 570 nm subtraction)
   in a Multiskan SkyHigh plate reader (Thermo Fisher Scientific, Waltham,
   MA, USA). Similarly, cellular proteome and secretome were determined in
   mouse and human chondrocytes by qualitative shotgun data-dependent
   acquisition (DDA) MALDI-TOF method [[156]86,[157]87,[158]88,[159]89].
   The DDA was performed in a micro-liquid chromatography (mLC) mass
   spectrometry (MS)/MS attached to a hybrid quadrupole-TOF/triple-TOF
   6600 and output data was filtered by 5% false discovery rate (FDR) and
   p ≤ 0.05 [[160]89]. The ortholog migration algorithm from REACTOME
   conversed mouse and human proteomes [[161]90]. The Venn diagram and
   interactome from FunRich software analyzed protein profiles [[162]91].
   The molecular function and pathway enrichment from REACTOME identified
   different medical and biological associations [[163]90].

2.8. Statistical Analysis and Sample Size

   The sample size (n) of the whole study was established in 6 independent
   biological replicas from different patients or cell passes and 3
   dependent technical replicas. The availability of non-primary cells of
   early pass was unlimited. On the contrary, the availability of primary
   cells isolated from OA patients or healthy corpses was very limited.
   Patients were excluded from the study (CAEIG-2016/258) if they were
   diagnosed with microcrystalline arthritis or any other infectious
   process. Moreover, even if they were not diagnosed, a further analysis
   of the fresh sample was also needed. Similarly, the presence of any
   other pathology apart from OA directly excluded those samples. All
   included samples presented sufficient cartilage and bone to perform the
   cell isolation. Nevertheless, only 3 healthy samples and 3 OA samples
   presented sufficient synovium to be removed and processed to isolate
   synoviocytes. Biochemical assays (Griess and MTT) required 10^4 cells
   (P96 plate), transcriptomics (RT-qPCRs) required 10^5 cells (P12),
   metabolic assays required 3 × 10^5 cells (P6) and proteomics (ELISA and
   MALDI-TOF) required 3.5 × 10^6 cells (P100) per data point. Overall,
   the use of primary cells was optimized to acquire the largest amount of
   data points. MALDI-TOF proteomics analysis was the most cell-demanding
   technique and, considering that thousands of technical replicas runs
   are performed, we optimized its sample size to 3 independent biological
   replicas.

   All datasets were expressed as the mean ± standard error mean (SEM) and
   normalized by the “Control” treatment data. Gene expression (mRNA) data
   was obtained by the RT-qPCR threshold cycle (ΔΔCt), also known as the
   ΔΔ quantification cycle (ΔΔCq). Gene expression was normalized by
   reference gene HPRT1 and analyzed by comparative transcriptomics.
   Fold-change (log[2]) units were used in the proteome heatmaps or column
   charts.

   The whole manuscript used The New England Journal of Medicine (NEJM)’s
   statistical significance method of representation. Specifically, a
   p-value of: <0.001 (***), <0.002 (**), <0.033 (*), and <0.12 (blank
   space or ns). Data distribution (Shapiro–Wilk and Kolmogorov–Smirnov
   normality tests) determined the use of parametric or non-parametric
   tests. Data from primary human cells were analyzed by non-parametric
   Kruskal–Wallis or Mann–Whitney test if needed. Otherwise, data were
   analyzed by parametric One Way-ANOVA and Tukey post-test or unpaired
   t-test Welch’s correction if needed.

3. Results

3.1. Determining the Proteome Profile of TLR4/IL1R-Stimulated Primary Human
OA Chondrocytes

   Anti-OA properties have been associated with BSE mix through NFκB
   pathway inhibition; nonetheless, the results were not consistent
   enough. Consequently, no international organizations such as the Food
   and Drug Administration (FDA) or the European Medicines Agency (EMA)
   have approved BSE mix or any individual component as a drug for
   clinical use. As such, our study focused on three pillars: the BSE mix
   single element BBA, the use of a primary joint cell model, and the
   implementation of novel techniques such as computational chemistry.

   Potential BBA targets described in the literature are downstream of the
   TLR4/IL1R signaling pathway, including NFκB. In this sense, we analyzed
   the secretome and proteome of TLR4/IL1R-activated human OA chondrocytes
   (hOC) isolated from OA patients to validate novel and already described
   inhibitory targets ([164]Figure 1).

Figure 1.

   [165]Figure 1
   [166]Open in a new tab

   Determining the human secretome and proteome of TLR4/IL1R-activated OA
   chondrocytes: (A–J) Secretome and proteome profile by DDA MALDI-TOFF of
   human OA chondrocytes stimulated with LPS (100 ng/mL) or IL1β (0.1
   ng/mL) for 48 h. Independent biological replicas (n = 3) were
   normalized (control), expressed as the mean and /or fold-change (FC);
   (A) human secretome profile of non-filtered (FDR ≤ 0% = 556) and
   filtered (FDR ≤ 5% = 201) secreted proteins; (B) Venn diagram (FDR ≤
   5%) of TLR4 (LPS)/IL1R (IL1β)-activated and non-activated (Control)
   human OA chondrocytes secretome; (C) interactome plot (FDR ≤ 5%) of
   TLR4/IL1R-activated human OA chondrocytes secretome; (D,E) enrichment
   analysis of molecular functions (FDR ≤ 5%; fold-change) in the
   TLR4/IL1R-activated human OA chondrocytes secretome; (F) human proteome
   profile of non-filtered (FDR ≤ 0% = 2804) and filtered (FDR ≤ 5% =
   2255) cellular proteins; (G) Venn diagram (FDR ≤ 5%) of TLR4 (LPS)/IL1R
   (IL1β)-activated and non-activated (Control) human OA chondrocytes
   proteome; (H) interactome plot (FDR ≤ 5%) of TLR4/IL1R-activated human
   OA chondrocytes proteome; (I,J) pathway enrichment analysis (FDR ≤ 5%;
   fold-change) ofTLR4/IL1R-activated human OA chondrocytes proteome.

   The secretome profile (FDR ≤ 5%) of hOC stimulated for 48 h with the
   TLR4 agonist LPS (100 ng/mL) and IL1R agonist IL1β (0.1 ng/mL])
   displayed 201 unique proteins ([167]Figure 1A). Multiple proteins were
   consistently expressed, whereas others were modulated after TLR4 (down
   48/up 35 proteins) and IL1R (down 52/up 26 proteins) activation
   ([168]Figure 1B). Interestingly, the interactome analysis showed that
   the most critical and highly associated proteins in TLR4/IL1R-activated
   hOC were β-actin (ACTB), MMP1, MMP3, and IL6 among others ([169]Figure
   1C). Furthermore, enrichment analysis of molecular functions in TLR4
   and IL1R-activated hOC identified multiple categories associated with
   anabolism depleted whereas the ones associated with catabolism were
   enriched ([170]Figure 1D,E).

   The human proteome profile (FDR ≤ 5%) displayed 2255 unique cellular
   proteins ([171]Figure 1F). Among them, multiple proteins were
   associated with TLR4 (down 234/up 292 proteins)/IL1R (down 636/up 137
   proteins) activation ([172]Figure 1G). At the centermost of the
   TLR4/IL1R-activated interactome plot, TLR4, Signal Transducer and
   Activator Transcription 3 (STAT3), NFKB1, MMP1, MMP3, IL1R, IL6, Tumor
   Necrosis Factor Receptor Associated Factor 6 (TRAF6), VCAM1, and MAPK14
   proteins were found among others ([173]Figure 1H). The similarity of
   results between TLR4 and IL1R-activated human proteomes was further
   observed in the pathway enrichment analysis of REACTOME innate immunity
   ([174]Figure 1I,J). Interestingly, TLR4/IL1R downstream signaling
   pathways MAPK/p38, NFκB, tumor necrosis factor (TNF), Interferon αβ
   (IFNαβ), ROS, and NLRP3 were found enriched ([175]Figure 1I,J).
   Contrarily, we found depleted extracellular matrix (ECM) synthesis and
   maintenance pathways ([176]Figure 1I,J).

   The use of animal or cellular mouse models is essential in
   translational medicine research as an initial step due to the scarcity
   of primary human OA experimentation. In this sense, we replicated the
   cellular proteome analysis workflow in TLR4/IL1R-activated mouse ATDC5
   chondrocytes ([177]Figure S1A–D). Interestingly, the mouse proteome
   profile identified a similar pattern of induced and inhibited proteins
   to the human proteome profile ([178]Figure S1A,B). Moreover, both TLR4
   and IL1R-activated mouse ATDC5 chondrocytes put at the centermost of
   the interactome ([179]Figure S1C) the pleiotropic Polyubiquitin C
   (Ubc), a protein known to promote NFκB pathway activation [[180]92]. In
   line with this, categories associated with MAPK/p38, NFκB, and ROS were
   found enriched among others, whereas ECM pathways were found depleted
   ([181]Figure S1D,E).

3.2. Predicting the Binding Affinity of β Boswellic Acid to Potential
Proteome Profile Targets

   Secretome and proteome pointed to NFκB, MAPK/p38 (MAPK14), TLR4, IL1R,
   and NLRP3 as the most promising targets for BBA inhibitory properties.
   The first three develop their activity in the cytosol; therefore, BBA
   needs to cross the cell membrane to perform inhibitory effects, whereas
   the others are innate immune receptors bound to a membrane. In this
   sense, through computational chemistry we explored the binding affinity
   of β boswellic acid (BBA) towards these biological targets ([182]Figure
   2).

Figure 2.

   [183]Figure 2
   [184]Open in a new tab

   Predicting the binding affinity of β boswellic acid to biological
   targets: (A–F) The ability of ligands (BBA, Cmpd1, KBA, AKBA, Mygalin,
   Curcumin, TAK242/CLI-095, Methotrexate, T5626448, MCC950, and ADP) to
   dock targets (A) IKK, (B) NFkB, (C) TLR4, (D) IL1R1, (E) TLR3, and (F)
   NLRP3 was ranked (F) according to the predicted binding energy and
   displayed the best result (pose) in each analysis. The more negative
   energy, the stronger the interaction ligand-target is. The selected
   computational order ranks relative to the affinity; (G) BBA cell
   permeability (red dots) using a quantitative structure–activity
   relationships (QSAR) model trained with cell permeability data of 190
   compounds (blue dots) and obtaining a log P[e] = 5.38; (H) molecular
   dynamics (MD) trajectories (100 ns) and root means-square deviation
   (RMSD) were used to measure the structural dynamic stability of a
   ligand docked in a target binding cite.

   The development of NFκB signaling inhibitors such as the Cmpd1
   inhibitor uses the IKK as a prime target [[185]66]. This inhibitor
   binds to the IKKβ at the hinge loop connecting the N and C lobes, a
   region that recognizes the adenine within the adenosine triphosphate
   (ATP) [[186]66]. Our simulations ([187]Figure 2A,F) revealed that Cmpd1
   is located at that region with significant large energy (−59.91
   kcal/mol). On the contrary, 11-keto-β-boswellic acid (KBA, −18.11
   kcal/mol), acetyl-11-keto-β-boswellic acid (AKBA, −18.96 kcal/mol), or
   BBA (−13.88 kcal/mol) are anchored with less negative binding energy;
   that is, with a less intense interaction ([188]Figure 2A,F).
   Consequently, these data demonstrated that the observed activity for
   BBA and derivatives was not associated with the IKK-related NFκB
   inhibition mechanism. The unsuccessful interaction results motivated us
   to assess the interaction of BBA against NFκB using the Müller et al.
   NFκB p50 (1SVC) [[189]93] homodimer bound to DNA. We explored the whole
   protein surface, and the best-ranked poses were retained for MM/GBSA
   refinement. Interestingly, the simulations suggested that BBA binds
   NFκB with a strong interaction energy of −31.92 kcal/mol ([190]Figure
   2B,F).

   This successful result led us to study targets upstream of NFκB such as
   the human TLR4 complex. This complex is formed by two subunits of TLR4
   protein (Chain A and B) and two subunits of accessory molecule MD2
   [[191]94] ([192]Figure 2C,F). TLR4 is a promiscuous innate immune
   response (IIR) receptor capable of binding to a large diversity of host
   (DAMPs) and non-host (PAMPs) derived structures. Our molecular model
   for TLR4 was designed using the crystal deposited in the PDB with code
   3VQ1 [[193]95] ([194]Figure 2C,F) Interestingly, the binding affinity
   ([195]Figure 1A) of BBA towards the human TLR4 complex (−45.15 Kcal/mol
   for Chain A) was found energetically on par with the specific
   pharmacological TLR4 inhibitor CLI-095/TAK242 (−47.29 Kcal/mol).
   Furthermore, similar results were obtained for curcumin (−58.49
   Kcal/mol) and mygalin (−65.98 Kcal/mol), two other candidates as TLR4
   inhibitors for clinical practice ([196]Figure 2A,F). Moreover, a
   similar trend was observed for TLR4 complex Chain B ([197]Figure 2A,F).

   Considering that computational analysis ranked BBA-TLR4 as the most
   promising target for BBA, we decided to further assess two TLR4-related
   receptors namely the IL1R and TLR3 complexes ([198]Figure 2D–F). The
   binding affinity of IL1R-BBA (−4.12 Kcal/mol) was found lower than
   IL1R-Methotrexate (−34.54 Kcal/mol), the main IL1R inhibitor
   ([199]Figure 2D,F). The TLR3 shares with the TLR4 part of the signaling
   cascade but it is activated by dsDNA and inactivated by inhibitors such
   as T5626448 (−12.19 Kcal/mol); nevertheless, the binding affinity
   TLR3-BBA (+2.18 Kcal/mol) was found inexistent ([200]Figure 2E,F).
   Finally, we addressed the potential affinity NLRP3-BBA, a signaling
   pathway downstream to TLR4 and IL1R. We compared the binding energy of
   the best docking pose predicted for BBA against the NLRP3 agonist ADP
   and antagonist MCC950 [[201]96]. According to the positive binding
   energy shown in our simulations, BBA (+7.35 kcal/mol) does not fit into
   that position, while both MCC950 (−69.80 kcal/mol) and ADP (−31.31
   kcal/mol) efficiently dock to the binding site ([202]Figure 2F).

   These accumulated results evidenced a predilection of BBA to bind the
   receptor TLR4; nonetheless, they do not exclude the possibility of
   directly interacting with NFκB after crossing the cellular membrane. We
   used a 190-compound dataset to train [[203]97] (training blue points)
   the Quantitative Structure Activity Relationships (QSAR) [[204]98]
   software to predict the effective drug cell permeability coefficient
   (log P[e]). Indeed, trained QSAR predicted (testing red points) an
   effective permeability coefficient of BBA (log P[e] = 5.38) that
   matches the naloxone permeability, a drug known to cross the cell
   membrane but also capable of binding and inhibiting the TLR4 signaling
   [[205]23] ([206]Figure 2G).

   This potential dual dynamic is interesting and not yet documented by
   the literature; onwards, we focused on the BBA binding effect on the
   TLR4 complex. In fact, we performed molecular dynamics (MD) simulations
   and root means-square deviation (RMSD) monitoring to test BBA-TLR4
   docking strength against the CLI-095-TLR4 complex ([207]Figure 2H).
   Specifically, RMSD values reveal that BBA (red line) is stabilized with
   an RMSD variation of 3 Å after 20 ns, whereas the reference inhibitor
   TAK242 required a larger RMSD window of 5 Å after 40 ns ([208]Figure
   2H). The smaller RMSD variation associated with BBA confirms the
   stability of the complex under biological conditions. Consequently, MD
   simulations ensured dynamic binding stability (100 ns trajectory) and
   RMSD monitoring ensured structural stability through a key TLR4 residue
   (Arg90) comparable for both BBA-TLR4 and CLI-095-TLR4 ([209]Figure 2H).

   Altogether, computational pharmacology found the TLR4 complex as the
   most probable target with large binding interaction energy comparable
   to the value predicted for one of the golden standard inhibitors,
   namely, CLI-095 or TAK242.

3.3. BBA Effects on Cell Viability and ROS Secretion in Chondrocytes

   Computational docking predicted a higher binding affinity of BBA
   towards the human TLR4 complex. This BBA feature was compatible with
   TLR4 antagonism. Moreover, BBA was predicted not to bind the IL1R
   complex. Previous studies reported that TLR4 blockade inhibited both
   TLR4 and IL1R signaling [[210]22]. Thus, we validated the in silico
   results of BBA actions in an in vitro model to determine its potential
   modulation of the TLR4/IL1R signaling cascade.

   Among the different pathways associated with TLR4/IL1R signaling, ROS
   generation was found highly enriched in the human and mice proteome
   profiles ([211]Figure 2I,J and [212]Figure S1D,E). Specifically, NO
   generation was a common denominator between TLR4 and IL1R activation
   ([213]Figure 2I,J and [214]Figure S1D,E). In fact, NO has been
   associated with the development of OA, the promotion of innate immune
   responses, the increase of articular catabolism, and the generation of
   more ROS ([215]Figure 3A) [[216]99,[217]100,[218]101]. Before
   proceeding to determine the effects of potential therapeutic BBA
   (0.10–5 µM) doses on ROS generation, we compared the basal innate
   immune response tone (mRNA) between primary human chondrocytes isolated
   from OA patients or healthy corpses ([219]Figure 3B). Primary human OA
   chondrocytes (hOC) presented higher mRNA levels for all the studied IIR
   factors ([220]Figure 3B). Furthermore, OA joint cells were found more
   sensitive to TLR4/IL1R activation than healthy ones ([221]Figure 3B).
   Next, we evaluated the balance between cell viability, the accumulation
   of NO subproduct NO[2-], and NOS2 gene expression (mRNA) to determine
   the effects of potential therapeutic BBA (0.10–5 µM) doses on ROS
   generation in primary hOC ([222]Figure 3C,D) as well as in mice ATDC5
   chondrocytes ([223]Figure S1F–I).

Figure 3.

   [224]Figure 3
   [225]Open in a new tab

   BBA effects on TLR4/IL1R-mediated innate immune response in primary
   human OA and healthy chondrocytes: (A) diagram of the interconnection
   between Osteoarthritis (OA) and TLR4/IL1R signaling, NOS activity,
   Reactive Oxygen Species (ROS) as well as Innate Immune Responses (IIR);
   (B–B’) primary human healthy (B) or OA (B–B’) chondrocytes (hOC) were
   treated with BBA (0.10–5 µM), CLI-095 (1 µM), or siTLR4 (15 nM) for 24
   h and cotreated with LPS (100 ng/mL) or IL1β (0.1 ng/mL) for another 48
   h. Independent biological replicas (n = 6) were normalized (control),
   expressed as the mean ±SEM and represented by the statistical
   significance NEJM system as *** (p-value < 0.001), ** (p-value <
   0.002), * (p-value < 0.033); (C) MTT cell viability assay in hOC
   chondrocytes displaying cell inviable doses (red), cell-viable doses
   (green) and FBS as a positive control; (B,D–I,M–B’) RT-qPCR gene
   expression (mRNA) of NOS2, TLR4, IL1R1, HSP90AA1, MAPK14/p38, LCN2,
   COX1, COX2, ICAM1, VCAM1, MCP1, NAMPT, MMP-1, -3, -9, -13, ADAMTS4,
   and/or ACAN and COL2α1 in primary human healthy or OA chondrocytes;
   (J–K) ELISA of secreted (J) or cellular (K) IL6 protein (pg/mL) in
   primary hOC; (L) Green malachite assay (Relative O.D. 460 nm) of
   phosphoproteome status in primary hOC.

   After LPS (100 ng/mL) and IL1β (0.1 ng/mL) stimulation, cell viability
   was unaltered ([226]Figure 3B,C and [227]Figure S1F,G) and accumulation
   of NO[2]^− was increased ([228]Figure S1H,I). Interestingly, unlike
   high concentrations of BBA [5 µM], cotreatment with BBA (0.10–1 µM) for
   48 h decreased NO[2-] accumulation in a dose-response manner without
   altering cell viability ([229]Figure 3B,C and [230]Figure S1F–I). Next,
   we evaluated BBA (0.10–1 µM) effects on NOS2 gene expression, also
   known as inducible nitric oxide synthase (iNOS). Results were
   consistent with ROS-NO[2] determination; activation of TLR4/IL1R
   increased NOS2 mRNA levels and BBA (0.10–1 µM) decreased them in a
   dose-response manner ([231]Figure 3D and [232]Figure S1J).

   Considering these results, we evaluated whether BBA effects were bound
   to the modulation of the expression of TLR4 or IL1R mRNA levels. Gene
   expression of TLR4 and IL1R showed an increase in mRNA levels after
   LPS/IL1β stimulation but no modulation after BBA co-treatment
   ([233]Figure 3E,F and [234]Figure S1K,L).

   Interestingly, treatment with the specific TLR4 small-molecule
   inhibitor CLI-095 (1 µM) or a siRNA anti-TLR4 (siTLR4) (15 nM)
   recapitulated BBA treatment effects ([235]Figure 3C–F and [236]Figure
   S1J–L). Furthermore, siTLR4 silencing treatment severely downregulated
   TLR4 mRNA levels but did not alter IL1R mRNA levels ([237]Figure 3E,F
   and [238]Figure S1K,L). Moreover, the blockade of the TLR4 complex by
   CLI-095 treatment did not alter TLR4 nor IL1R mRNA levels ([239]Figure
   3E,F and [240]Figure S1K,L).

3.4. BBA Effects on TLR4/IL1R-Mediated IIRs in Primary Human OA Chondrocytes

   Once the inhibitory effects of BBA in the NOS2 axis were demonstrated,
   we further explored other TLR4/IL1R-modulated pathways found enriched
   in the proteome analysis including MAPK p38/NFκB, IFNαβ, and TNF
   ([241]Figure 1; [242]Supplementary data S2). The MyD88-dependent MAPK
   p38/NFκB downstream pathway is central to TLR4/IL1R signaling. We
   evaluated the mRNA expression of common specific elements in the MAPK
   p38 and NFκB pathway, namely, HSP90AA1, MAPK14 or p38, and IL6 in
   primary human OA chondrocytes (hOC). We found increased mRNA levels of
   HSP90AA1, p38, and IL6 after TLR4/IL1R activation ([243]Figure 3G–I)
   and decreased ones after cotreatment with BBA (0.10–1 µM), CLI-095 (1
   µM), and siTLR4 (1 µM) in a dose-response dynamic ([244]Figure 3G–I).
   To validate these data, we further tested the intracellular and
   secreted levels of IL6 in these cells. The TLR4/IL1R activation
   increased secreted ([245]Figure 3J) and cellular ([246]Figure 3K)
   protein concentration of IL6 (up to 584 pg/mL) while BBA and CLI-095
   cotreatment inhibited it. Consistent with a decreased activation of
   MAPK p38/NFκB pathway by BBA, we observed in TLR4/IL1R-activated hOC a
   reduction in the total phosphoproteome after BBA treatment ([247]Figure
   3L). Interestingly, cotreatment with BBA or CLI-095 also reduced the
   protein concentration of IL6 and phosphoproteome levels in
   non-stimulated cells ([248]Figure 3J–L).

   Next, we evaluated the TNF pathway element TNFα, the MyD88-independent
   IFNαβ pathway element IFNβ1, and highly studied innate immune response
   factors associated with the TLR4/IL1R pathway including MAPK p38/NFκB
   downstream signaling factors in the OA context. Remarkably, most of
   these factors were directly identified in the secretome and proteome
   profiles ([249]Figure 1A–J and [250]Figure S1A–E). At the
   transcriptomic level, inflammatory factors (IFNβ1, TNFα, LCN2, COX1,
   COX2, ICAM1, VCAM1, MCP1, and NAMPT) and catabolic factors (MMP1, MMP3,
   MMP9, MMP13, and ADAMTS4) mRNA levels were increased after LPS and IL1β
   stimulation of primary hOC ([251]Figure 3M–Z). In contrast, mRNA levels
   of anabolic factors (ACAN and COL2A1) were decreased after LPS and IL1β
   stimulation of primary hOC ([252]Figure 3A’–B’). Gene expression (mRNA)
   of inflammatory and catabolic factors decreased in a dose-response
   manner after cotreatment with BBA, CLI-095, or siTLR4 ([253]Figure
   3M–Z). In contrast, gene expression (mRNA) of anabolic factors was
   increased and reverted to basal control levels after BBA, CLI-095, or
   siTLR4 cotreatment ([254]Figure 3A’–B’). Similar results were obtained
   in mice ATDC5 chondrocytes ([255]Figure S1M,N and [256]Figure S2A–O).

3.5. BBA Effects on TLR4/IL1R-Mediated IIRs in Primary Human OA Osteoblasts

   The OA disease progression begins degrading the cartilage and
   eventually alters the bone structure ([257]Figure 4A). As such, we
   evaluated the BBA inhibitory effects on the TLR4/IL1R downstream
   pathways in primary human OA osteoblasts (hOB) isolated from OA
   patients. Remarkably, primary hOB presented higher mRNA levels for all
   the studied IIR factors. Similarly, hOB was more sensitive to TLR4/IL1R
   activation than healthy ones ([258]Figure 4B). In this sense, we used
   BBA (0.10–1 µM), CLI-095 (1 µM), and siTLR4 (1 µM) doses that did not
   alter primary hOB cell viability ([259]Figure 4B–A’).

Figure 4.

   [260]Figure 4
   [261]Open in a new tab

   BBA effects on TLR4/IL1R-mediated innate immune response in primary
   human OA and healthy osteoblasts: (A) diagram of the interconnection
   between articular tissues in osteoarthritis; (B–A’) primary human
   healthy (B) or OA (B–A’) osteoblast (hOB) were treated with BBA (0.10–5
   µM), CLI-095 (1 µM) or siTLR4 (15 nM) for 24 h and cotreated with LPS
   (100 ng/mL) or IL1β (0.1 ng/mL) for another 48 h. Independent
   biological replicas (n = 6) were normalized (control), expressed as the
   mean ± SEM and represented by the statistical significance NEJM system
   as *** (p-value < 0.001), ** (p-value < 0.002), * (p-value < 0.033);
   (C) MTT cell viability assay in hOB osteoblasts displaying cell
   inviable doses (red), cell-viable doses (green) and FBS as a positive
   control; (B,D–I,M–A’) RT-qPCR gene expression (mRNA) of NOS2, TLR4,
   IL1R1, HSP90AA1, MAPK14/p38, LCN2, COX1, COX2, ICAM1, VCAM1, MCP1,
   NAMPT, MMP-1, -3, -9, -13, ADAMTS4, and/or COL1A1 in primary human
   healthy or OA osteoblasts; (J,K) ELISA of secreted (J) or cellular (K)
   IL6 protein (pg/mL) in primary hOB; (L) Green malachite assay (Relative
   O.D. 460 nm) of phosphoproteome status in primary hOB.

   Interestingly, ROS/NOS2 and MAPK p38/NFκB pathway ([262]Figure 4D–L)
   presented a similar transcriptomic profile to hOC chondrocytes
   ([263]Figure 3D–L). The 48-h stimulation with LPS and IL1β increased
   NOS2, TLR4, IL1R, HSP90α, p38, and IL6 mRNA levels, as well as IL6
   protein levels and the phosphoproteome status ([264]Figure 4D–L). In
   line with hOC results, BBA, CLI-095, and siTLR4 severely reduced the
   expression levels of all the studied factors except IL1R ([265]Figure
   4D–L). Besides siTLR4, neither BBA nor CLI-095 inhibited TLR4 mRNA
   levels ([266]Figure 4E).

   The transcriptomic profile of the other TLR4/IL1R downstream signaling
   factors including TNF and IFNαβ pathways in primary hOB osteoblasts was
   similar to the hOC chondrocytes. As such, stimulation with LPS and IL1β
   in primary hOB increased inflammatory factors (IFNβ1, TNFα, LCN2, COX1,
   COX2, ICAM1, VCAM1, MCP1, and NAMPT) and catabolic factors (MMP1, MMP3,
   MMP9, MMP13, and ADAMTS4) mRNA levels but decreased anabolic factor
   (COL1A1) mRNA levels ([267]Figure 4M–A’). Consequently, BBA, CLI-095,
   or siTLR4 cotreatment increased anabolic factors to basal levels and
   decreased inflammatory and catabolic factors gene expression in a
   dose-response manner ([268]Figure 4M–A’). Overall, the intensity of
   TLR4/IL1R-activation on hOB osteoblasts mRNA and protein levels was
   lower compared to hOC chondrocytes; nevertheless, BBA, CLI-095, or
   siTLR4 co-treatment effects were the same ([269]Figure 3 and
   [270]Figure 4).

3.6. BBA Effects on TLR4/IL1R-Mediated IIRs in Human OA Synoviocytes

   The pathophysiology from early-to-late OA regularly causes inflammation
   of the synovium; in turn, inflamed synovia promotes the alteration of
   other joint tissues ([271]Figure 5A). Consequently, we evaluated the
   BBA inhibitory effects in human synoviocytes. In this sense, primary
   human OA synoviocytes (hOS) presented higher mRNA levels for all
   TLR4/IL1R-associated innate immune factors ([272]Figure 5B). In fact,
   hOS were more sensitive to TLR4/IL1R activation than healthy ones
   ([273]Figure 5B). Nevertheless, the scarcity of primary human OA
   synoviocytes forced us to switch to human SW982 synoviocytes (hSW).
   Notably, the evaluation of BBA inhibitory effects on the TLR4/IL1R
   downstream pathways did not use doses of BBA (0.10–1 µM), CLI-095 (1
   µM), and siTLR4 (1 µM) that altered hSW cell viability ([274]Figure
   5C–Z).

Figure 5.

   [275]Figure 5
   [276]Open in a new tab

   BBA effects on TLR4/IL1R-mediated innate immune response in human
   synoviocytes: (A) Diagram of the interconnection between articular
   tissues in osteoarthritis; (B–Z) primary human healthy (B), OA (B) and
   non-primary human SW982 (C–Z) synoviocytes (hSW) were treated with BBA
   (0.10–5 µM), CLI-095 (1 µM), or siTLR4 (15 nM) for 24 h and cotreated
   with LPS (100 ng/mL) or IL1β (0.1 ng/mL) for another 48 h. Independent
   biological replicas (n = 6) were normalized (control), expressed as the
   mean ±SEM and represented by the statistical significance NEJM system
   as *** (p-value < 0.001), ** (p-value < 0.002), * (p-value < 0.033);
   (C) MTT cell viability assay in hSW synoviocytes displaying cell
   inviable doses (red), cell-viable doses (green) and FBS as a positive
   control; (B,D–I,M–Z) RT-qPCR gene expression (mRNA) of NOS2, TLR4,
   IL1R1, HSP90AA1, MAPK14/p38, LCN2, COX1, COX2, ICAM1, VCAM1, MCP1,
   NAMPT, MMP-1, -3, -9, -13, and ADAMTS4 in human healthy (B), OA (B) and
   hSW synoviocytes (D–I,M–Z); (J,K) ELISA of secreted (J) or cellular (K)
   IL6 protein (pg/mL) in hSW synoviocytes; (L) Green malachite assay
   (Relative O.D. 460 nm) of phosphoproteome status in hSW synoviocytes.

   The activation of TLR4/IL1R for 48 h in hSW synoviocytes modulated the
   expression of all TLR4/IL1R-associated genes being studied ([277]Figure
   5D–Z). The innate immune factors (NOS2, TLR4, IL1R, HSP90α, p38, IL6,
   IFNβ1, TNFα, LCN2, COX1, COX2, ICAM1, VCAM1, MCP1, NAMPT, MMP1, MMP3,
   MMP9, MMP13, and ADAMTS4) were upregulated (mRNA). Similarly,
   TLR4/IL1R-activated hSW synoviocytes increased protein levels of
   secreted and cellular IL6 as well as the phosphoproteome status
   ([278]Figure 5J–L).

   The use of BBA, CLI-095, and siTLR4 downregulated (mRNA, protein, and
   phosphoproteome status) all factors except IL1R ([279]Figure 5D–Z).
   Likewise, neither BBA nor CLI-095 inhibited TLR4 mRNA levels, only the
   siTLR4 was capable ([280]Figure 5D–Z). Overall, the intensity of
   TLR4/IL1R-activation on hSW synoviocytes mRNA and protein levels was
   higher compared to hOC chondrocytes and hOB osteoblasts. Nonetheless,
   BBA, CLI-095, or siTLR4 co-treatment effects were very similar in the
   three human joint cells: chondrocytes ([281]Figure 3), osteoblasts
   ([282]Figure 4), and synoviocytes ([283]Figure 5).

3.7. BBA Effects in Joint Cells on the NLRP3 Inflammasome Pathway

   The proteome enrichment analysis of TLR4/IL1R-activated primary human
   OA chondrocytes identified specific pathways downstream of these
   receptors but upstream of relevant key factors such as MAPK p38 or
   NFκB. The transcriptomic and protein analysis further supported that
   these pathways could contribute to the mechanism of action of BBA
   inhibitory effects. Remarkably, the proteome profile identified the
   NLRP3 inflammasome signaling as a pathway enriched after the activation
   of TLR4 and IL1R ([284]Figure 1 and [285]Figure S1). Furthermore, the
   activation of the NLRP3 pathway triggers CASP1 that catalyzes the
   transformation of pro-IL1β into mature IL1β, the canonical agonist of
   IL1R1.

   As such, we decided to further explore this potential mechanism of
   action in primary human OA joint cells. In TLR4/IL1R-activated hOC,
   gene expression (mRNA) of NLRP3, CASP1, and IL1β was highly increased
   ([286]Figure 6A–C). In contrast, treatment with BBA, CLI-095, or siTLR4
   reduced the induced mRNA levels ([287]Figure 6A–C). Interestingly,
   NLRP3 silencing with siRNA (siNLRP3) recapitulated the blocking effects
   of BBA, CLI-095, and siTLR4 in TLR4/IL1R-activated hOC ([288]Figure
   6A–C). Furthermore, CASP1 activity assay and IL1β ELISA protein assay
   were used to validate these results ([289]Figure 6D–F). Stimulation
   with LPS or IL1β increased CASP1 activity and cellular and secreted
   IL1β protein ([290]Figure 6D–F). In line with previous results,
   treatment with BBA and CLI-095 reduced CASP1 activity and cellular and
   secreted IL1β levels to basal levels ([291]Figure 6D–F).

Figure 6.

   [292]Figure 6
   [293]Open in a new tab

   BBA effects on NLRP3-mediated innate immune response in primary joint
   cells: (A–R) Primary human OA chondrocytes (hOC), primary human OA
   osteoblasts (hOB), and human SW982 synoviocytes (hSW) were treated with
   BBA (1 µM), CLI-095 (1 µM), siTLR4 (15 nM) and siNRLP3 (15 nM) for 24 h
   and cotreated with LPS (100 ng/mL) or IL1β (0.1 ng/mL) for another 48
   h. Independent biological replicas (n = 6) were normalized (control),
   expressed as the mean ± SEM, and represented by the statistical
   significance NEJM system as *** (p-value < 0.001), ** (p-value <
   0.002), * (p-value < 0.033); (A–C,G–I,M–O) RT-qPCR gene expression
   (mRNA) of NLRP3, CASP1, and IL1β in hOC chondrocytes (A–C), hOB
   osteoblasts (G–I), and hSW synoviocytes (M–O); (D,J,P): CASP1 activity
   (pNA reaction) assay in hOC chondrocytes (D), hOB osteoblasts (J), and
   hSW synoviocytes (P); (E,F,K,L,Q,R) ELISA of cellular (E,K,Q) and
   secreted (F,L,R) IL1β protein (pg/mL) in hOC chondrocytes (E,F), hOB
   osteoblasts (K,L) and hSW synoviocytes (Q,R).

   The NLRP3 pathway plays a relevant role in bone differentiation and
   inflammation [[294]102]. The TLR4/IL1R activation of hOB increased mRNA
   levels of NLRP3, CASP1, and IL1β ([295]Figure 6G–I). Moreover, this
   activation also increased CASP1 activity and cellular and secreted IL1β
   protein levels ([296]Figure 6J–L). As observed in hOC, treatment with
   TLR4 inhibitors BBA, CLI-095, or siTLR4, decreased NLRP3 expression, as
   well as CASP1 activity and IL1β protein levels ([297]Figure 6G–L).
   Remarkably, siNLRP3 recapitulated the effects of TLR4 inhibition by
   reducing the mRNA levels of NLRP3, CASP1, and IL1β ([298]Figure 6G–L).
   Likewise, the NLRP3 pathway is critical for synovial inflammation.
   Consistent with this, TLR4/IL1R-activated hSW synoviocytes exhibited
   increased mRNA levels of NLRP3, CASP1, and IL1β, increased CASP1
   activity, and increased cellular and secreted IL1β levels ([299]Figure
   6M–R). Treatment with TLR4 inhibitors BBA, CLI-095, siTLR4, and/or
   siNLRP3 reduced all the studied NLRP3 pathway elements ([300]Figure
   6M–R). Interestingly, these effects were also observed in non-activated
   joint cells (chondrocytes, osteoblasts, and synoviocytes) ([301]Figure
   6A–C).

   All in all, the mechanism of action of BBA inhibitory effects on
   TLR4/IL1R-mediated IIR has proven to be directly associated with the
   NLRP3 pathway as well as MAPK p38 and NFκB, IFN, and TNF pathways
   ([302]Figure 7). Furthermore, BBA inhibitory effects might also come
   from the partial interruption of the positive feedback between IIR
   factors, ROS, DAMPs/PAMPs, and the TLR4/IL1R crosstalk ([303]Figure 7).

Figure 7.

   [304]Figure 7
   [305]Open in a new tab

   Diagram of TLR4/IL1R signaling, MAPK p38/NFκB, IFN, TNF, and NLRP3
   downstream pathways and innate immune response (IIR) in osteoarthritis
   (OA) joint cells.

4. Discussion

   We present evidence that β boswellic acid or 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, we determined in stimulated and
   non-stimulated articular cells that TLR4 inhibition by BBA or other
   known inhibitors is associated with NLRP3 downregulation. Moreover, we
   revealed that this downregulation was enough to recapitulate the
   effects of BBA or TLR4 inhibition on TLR4/lL1R-mediated IIR.
   Altogether, we provided relevant data supporting BBA as a strong TLR4
   inhibitor that may help to control OA-associated IIR.

   The BBA is extracted from Boswellia serrata and it is present in the
   BSE mix of triterpenes, essential oils, and polysaccharides that it is
   commonly taken as an infusion or food complement in traditional
   ayurvedic medicine to treat inflammatory diseases such as asthma,
   inflammatory bowel disease, RA, and OA. Interestingly, the evidence
   failed to demonstrate that BSE anti-inflammatory effects were a
   consequence of 5-lipoxygenase [[306]27] inhibition. On the contrary,
   the BSE element BBA has recently been described to inhibit NFκB, a
   downstream signaling pathway of TLR4/IL1R [[307]103,[308]104].
   Remarkably, TLR4 and IL1R are two innate immune receptors associated
   with the development of multiple rheumatic diseases
   [[309]17,[310]38,[311]39,[312]40,[313]41,[314]50,[315]51,[316]52]. The
   IL1R has been used as a therapeutic target for diseases such as OA
   [[317]17], RA [[318]50], or gout [[319]105]. However, there is no
   clinically available TLR4 inhibitor to treat rheumatic inflammatory
   diseases [[320]22,[321]106]. Considering the traditional use of BSE as
   antirheumatic and the new potential properties of BBA, we decided to
   determine whether BBA was able to block TLR4 and inhibit OA-associated
   IIR in joint cells.

   Considering that a functional TLR4 receptor is required for full IL1R
   signaling, the first approach was to analyze the primary human OA
   chondrocytes secretome and proteome ([322]Figure 1) changes linked to
   TLR4 and IL1R activation [[323]22]. We obtained key data on the
   TLR4/IL1R downstream signaling pathways. Specifically, MAPK p38/NFκB,
   TNF, IFNαβ, ROS, and NLRP3 downstream pathways were found enriched
   whereas ECM anabolic pathways were found depleted in the proteome of
   stimulated chondrocytes. These proteomic changes were consistent with
   increased inflammatory and catabolic OA-like phenotype
   [[324]107,[325]108,[326]109]. Likewise, the enrichment in ROS-related
   pathways including NO synthesis was in concordance with OA worsening
   associated with oxidative stress [[327]110,[328]111]. Similar results
   were obtained in mice ATDC5 chondrocytes proteome ([329]Figure S1).

   Next, through computational pharmacology, we investigated BBA potential
   inhibition of key factors implicated in major pathways found in the
   secretome and proteome of TLR4/IL1R-activated primary human OA
   chondrocytes ([330]Figure 2). We predicted a greater binding affinity
   of BBA to the human TLR4 complex than other elements suggested in the
   literature such as NFκB [[331]93] and IKK [[332]66,[333]112].
   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, as the literature
   suggested. Nonetheless, molecular dynamics simulations presented
   additional evidence of BBA-TLR4 binding stability, suggesting that
   specific TLR4 inhibition may be the main mechanism of action
   ([334]Figure 2).

   The combination of anti-TLR4 predicted activity of BBA and the
   TLR4/IL1R targets identified from the proteome analysis supported the
   in vitro study of BBA effects in TLR4/IL1R-activated human
   chondrocytes, osteoblasts, and synoviocytes. The relevance of this
   predicted BBA activity was underpinned by the observation of increased
   levels of OA-associated factors as well as increased sensitivity to
   TLR4 and IL1R agonists in chondrocytes, osteoblasts, and synoviocytes
   isolated from OA patients in contraposition to healthy ones.

   Data obtained in all these cells revealed that BBA cotreatment reduced
   the gene expression of factors related to MAPK p38/NFκB (IL6, LCN2,
   COX1, COX2, ICAM1, VCAM1, MCP1, and NAMPT), ECM catabolism (MMP-1, -3,
   -9, -13, and ADAMTS4), TNF, IFN, NLRP3 inflammasome (NLRP3, CASP1,
   IL1β), and ROS pathways in a dose-response manner. In contrast, BBA
   cotreatment restored the ECM anabolism loss after TLR/IL1R activation.
   Supporting these results, the protein expression of key cytokines and
   the phosphoproteome of these stimulated cells was significantly reduced
   by BBA anti-TLR4 effect. Furthermore, BBA activity did not cause
   negative effects on cell vitality. Similar results were obtained in
   mice ATDC5 chondrocytes.

   Considering that the specific TLR4 inhibitor CLI-095 and the anti-TLR4
   siRNA replicated the same BBA inhibitory effects in all the cellular
   studies [[335]113], and neither CLI-095 nor BBA downregulated TLR4 and
   IL1R expression, it was confirmed that TLR4 inhibition is the mechanism
   underlying BBA effect. In agreement with this, previous reports with
   other TLR4 inhibitors have obtained similar results about the key role
   of TLR4 inhibition on IL1R signaling [[336]22,[337]23]. Remarkably, BBA
   downregulated basal expression levels of several IIR factors suggesting
   that TLR4 constitutive activation contributes to the expression of
   multiple IIR factors in joint cells.

   Despite BBA not binding to to IL1R, it exhibited outstanding effects in
   blocking IL1R-mediated IIR. This was consistent with the described
   anti-inflammatory effects of TLR4 inhibition on the IL1-mediated RA
   animal model [[338]114]. The NLRP3 inflammasome pathway has been
   associated with the IIR in multiple rheumatic diseases
   [[339]115,[340]116], including OA [[341]117]. Moreover, this pathway
   was substantially enriched in chondrocytes upon the activation of TLR4
   and ILR1 receptors. In line with this, the NLRP3 inflammasome pathway
   has been identified downstream of TLR4/IL1R crosstalk
   [[342]117,[343]118]. Specifically, it participates in the synthesis and
   release of IL1β, which generates DAMPs that activate TLR4 [[344]17]
   promoting an inflammatory positive feedback loop. Accordingly, we
   investigated whether NLRP3 signaling pathway modulation could be
   responsible for BBA effects. Interestingly, in all the articular cells
   studied, BBA was able to downregulate the TLR/IL1R-induced expression
   of the NLRP3 axis (NLRP3, CASP1, and IL1β) as well as the enzymatic
   activity of CASP1, and the concentration of cellular and secreted IL1β
   protein. Similar results were obtained in non-activated OA joint cells.
   Consistent with this, NLRP3 and TLR4 silencing recapitulated the
   inhibitory effects of BBA and CLI-095 in OA joint cells. These data
   demonstrated that NLRPL3 signaling is tightly related to TLR4 and
   confirmed that BBA effects on IL1R signaling are TLR4- and
   NLRP3-dependent. Overall, the local use of BBA on the articular tissues
   might be open to consideration.

5. Conclusions

   We demonstrate that BBA inhibits OA-associated inflammatory and
   catabolic processes in chondrocytes, osteoblasts, and synoviocytes
   without altering the joint cell viability. We prove that the specific
   binding of BBA to the TLR4 complex inhibits TLR4 and IL1R signaling and
   downregulates MAPK p38/NFκB, NLRP3 inflammasome, and other
   OA-associated pathways. Moreover, we identified the TLR4-NLRP3 axis as
   an underlying mechanism of the BBA effect on IL1R signaling.
   Altogether, we present a solid bulk of evidence that BBA blocks OA
   innate immune responses and could be transferred into the clinic as an
   alimentary supplement or as a therapeutic tool after clinical trial
   evaluations.

Acknowledgments