TLRs are expressed on the cell surface or intracellularly in many cell types, especially in cells of the innate immune system, where they act as sensors of infection or damage. Activating TLRs lead to the heightened expression of various inflammatory genes, which have a protective role against infection.
Data rising predominantly from human patients and animal models of autoimmune disease indicate that inappropriate triggering of TLR pathways by exogenous or endogenous ligands may cause the initiation and/or perpetuation of autoimmune reactions and tissue damage. It was reported that TLRs are expressed in the inflamed joints of patients with RA and play an important role in the progression and maintenance of the disease, supporting inflammation and cartilage and bone damage [27]. Here we evidenced that TLR9 is highly expressed in RASF cultured “in vitro” in synovial fluid, to reproduce the physiopathological environmental characteristic of RA joints. To date there are not many published studies investigating the expression and function of TLR9 in synovial fibroblasts, and it is not yet clarified how much synovial fibroblasts can contribute to immune response induced by DNA sensing [28]. Moreover, TLR9 is essential for recognition of microbial CpG DNA or synthetic CpG oligonucleotide analogues containing a CpG oligodeoxynucleotide (ODN) and TLR9 signalling is initiated by recruitment of the adaptor molecule MyD88 followed by the engagement of interleukin (IL)-1R-associated kinases and tumour necrosis factor alpha receptor (TNFR)-associated factor [29], inducing the early activation of NF-kB and production of cytokines such as TNFa [30].
In RA the inhibitory proteins of apoptosis (IAPs) play a fundamental role as are responsible for hyperplasia of synovial fibroblasts and pannus formation at the cartilage–bone interface, where it cloaks the cartilage and erodes the bone [31]. Recently, it has been observed that IAPs play a crucial role in regulation of genes involved in innate and immune immunity, inflammation, survival and cell migration [32]. In particular, the IAPs involvement in innate immunity depends on the modulation of TLRs pathways, that lead to NF-kB transcription, MAP kinase and JNK activation [33].
Smac mimetic compounds are able to induce apoptosis in cancer cells and, unexpectedly, to induce the degradation of IAPs, blocking TLRs activation signals [34]. Thus, we investigated the effect of BV6 on RASF, that are resistant to apoptosis, upon TLR9 activation and we demonstrate that BV6 induces apoptosis not only in RASF cultured in tissue medium but also when TLR9 is activated. Both mRNA and protein of cleaved caspase 3 and caspase 8 were observed in RASF treated with BV6, as well as the proteolytic activation of pro-caspase, to confirm the ability of BV6 to abrogate the IAPs-mediated caspase inhibition. Probably, BV6 induced rapid degradation of cIAP-1, cleavage of pro-caspase-3 and caspase-dependent cell apoptosis and activated the NF-κB pathway and potentially promoted caspase-dependent and TNFα-induced cell death synergistically [35]. Moreover, the treatment with BV6 inhibits the NF-κB canonical pathway by binding and degrading cIAPs and this prevents the ubiquitylation of RIPK1 and leads to the formation of a complex containing RIPK1, caspase 8, and FADD, which promotes apoptosis. [35,36]
There is a substantial evidence that IAPs block cell death induced by TLRs on RASF, but there are only a few reports on the role of IAPs in the regulation of TLR-induced cytokines production.
After TLRs ligation, RASF can secrete more mediators that contribute to the maintenance and perpetuation of the inflammation in RA [16, 37]. It is well known that pro-inflammatory cytokines produced by inflamed synovial tissues in RA such as TNF, IL1, IL6, and IL1, can affect RASF activity and the differentiation of osteoclasts and osteoblasts with effects on bone. In addition, anti-inflammatory cytokines, including IL10, are present in RA joints and inhibit pro-inflammatory cytokines [38,39]
Our results show that TLR9 agonist ODN is able to elicit responses mRNA and protein expression for TNFα, IL6 and IL15 and BV6 inhibits this effect while upregulate antinflammatory cytokine IL10 expression. In RA IL10 inhibits pattern recognition receptor signaling through mechanisms, which include downregulation of MyD88 expression [40], and the ubiquitination and subsequent degradation of MyD88-dependent signaling molecules such as TRAF6 [41].
Innate pathways can potentially activate viral and stress-inducible gene expression of chemokines and cytokines that promote inflammation, cell recruitment, and joint destruction in RA. These signaling pathways have been implicated in inflammatory arthritis, and the IFN signature induced by innate receptor activation has been observed in RA and in addition to the synovium, an IFN profile has been reported in peripheral blood cells of a subset of RA patients (17). One possibility
is that the IFN regulatory factor (IRF) family as IRF7 regulate the IFN response in RA [27,42,43]
In RASF untreated IRF7 is weakly express while in the cells treated with ODN we observed an upregulation of IRF7 expression. IRF7 is crucial to induce maximally the expression of IFN-α/β, indeed, the death domain of MyD88 interacting with IRF7 stimulates the IFN-a promoters and [44]. Our data support the hypothesis that BV6-induced apoptosis is potentiated in ODN-activated RASFs by IRF7 upregulation that leads to apoptosis through the TGF-β signal pathway [45,46].
Our data extend knowledge of BV6 and underline the multiple effects on RA of this compound which could make it a valid support for the therapy of diseases where several mediators are involved, such as RA, and in which the simultaneous action on several aspects can be a successful strategy.