Disease severity and articular damage are associated with increased risk for disability, joint deformities and reduced life expectancy in patients with RA [21–23]. Yet, little is known about the genes implicated in the regulation of disease severity and articular damage genes in RA, and these genes could be the most relevant targets for new therapies aimed at preserving the joint architecture and function.
We have previously identified Cia5a, a 20.6 Mb arthritis severity and joint damage regulatory locus, on rat chromosome 10 . Cia5a co-localizes with other arthritis severity loci identified in other rodent models of arthritis such as oil-induced arthritis (Oia3) , and CIA in a DAxACI intercross (Cia27) . There have been no genome-wide association or linkage studies of disease severity and joint damage in RA, and therefore, it is unknown whether the Cia5a syntenic region on human chromosome 17q22-q25 harbors a severity or joint damage arthritis regulatory gene. However, the human 17q22-q25 region contains a locus previously linked with RA susceptibility . In the present study we analyzed synovial tissues from DA rats, which develop severe arthritis (PIA) with pronounced synovial hyperplasia and cartilage and bone destruction, and synovial tissues from the DA.F344(Cia5a) congenics, which develop mild and non-erosive disease. These two strains share the same MHC and are genetically identical except for the presence of F344 alleles at the Cia5a interval, underscoring the magnitude of the effect of this single locus on clinical disease, on histologic joint damage  and on gene expression (present study). DA.F344(Cia5a) congenics had significantly reduced expression of genes previously implicated in RA pathogenesis, RA severity and articular damage, including Il1b, Il18, Mif, Mmp3 and Mmp14. These and other similarities between DA rats and RA synovial tissues’ gene expression, such as increased expression of chemokines, matrix proteins, adhesion molecules, mediators of innate immune responses, and others, underscore and further validate the potential clinical relevance of our model and discovery strategy.
We identified a new role for Cia5a on the regulation of the expression of members of the Syk pathway, where forty-seven genes directly or indirectly related to Syk activation were expressed in increased levels in DA, and significantly reduced levels in DA.F344(Cia5a) congenics. Syk is a tyrosine kinase that phosphorylates ITAM motifs in trans-membrane receptors or adaptors, and interacts with partners like Vav, PI3K and Slp76. Syk activation mediates signaling through several cell surface receptors, including those with significantly different levels in this study such C-lectin type receptors, Fcer1g, Fcgr2a, Trem2, Tyrobp, integrins, and the T-cell receptor (TCR) (Figure 3). Resident and infiltrating inflammatory cells in the RA synovial pannus, such as mast cells, macrophages, B and T cells, express these Syk-activating receptors. These resident cells and infiltrating cells have been implicated in arthritis pathogenesis and joint damage, raising the possibility that part of their effect may be mediated by Syk-activating receptors.
Analyses of cell-specific genes suggested reduced numbers of macrophages, dendritic cells, neutrophils and T cells in the synovial tissues of congenics compared with DA, which is in agreement with our previous histologic analyses and might explain part, but not all of the differences in expression of Syk genes. Additionally, DA.F344(Cia5a) congenics had significantly lower levels of the FLS-specific gene Cdh11, compatible with the reduced synovial hyperplasia that we have previously described.
Syk pathway members regulate several cellular processes implicated in arthritis pathogenesis and articular damage, ranging from the production of reactive oxygen species, NFκB activation and the transcription of pro-inflammatory mediators such as Il1b and Ccl2, to the cell proliferation required for the development of synovial hyperplasia, and actin cytoskeleton rearrangements . NFκB activity is regulated by Syk and by several other pathways including TLRs and cytokine receptors . The NFκB pathway has a central role in the production of pro-inflammatory cytokines such as IL-1β, IL-6 and TNFα, in the development of synovial hyperplasia and in disease severity [29–31]. Actin cytoskeleton rearrangements are also regulated by the Syk pathway , and are required for the migration of inflammatory cells into the synovial tissue, and for synovial cells and synovial tissue invasion and destruction of cartilage [13, 32]. Therefore, our observations suggest that a gene located within the Cia5a interval is a new regulator of the expression of Syk pathway genes implicated in key processes in arthritis pathogenesis.
The precise mechanisms whereby Cia5a regulates the expression of Syk genes remain unclear, and might reflect differences in tissue cellularity, multiple cell-activating processes, or a polymorphism in transcription factor located within the Cia5a interval that affects transcription. Studies by our group of synovial tissues obtained from four different congenic strains yielded different results in gene expression (Brenner et al., manuscript in preparation) [12, 14, 33], suggesting that the Syk-regulatory effect of Cia5a is a specific observation, and not simply related cellularity differences or inflammation.
Syk has been recently implicated in arthritis pathogenesis and joint damage, and Syk-deficient mice are protected from autoantibody-induced erosive arthritis , and treatment with a SYK inhibitor significantly reduced disease severity and joint erosions and damage in collagen-induced arthritis . Both the total and phosphorylated forms of SYK are expressed in increased levels in RA synovial tissues compared with osteoarthritis, and SYK inhibition reduced the expression of IL-6 and MMP-3 . More importantly, the use of a SYK inhibitor significantly reduced disease activity in patients with RA , with 67%, 43% and 28% of patients achieving ACR20, ACR50 and ACR70, respectively, in a 3-month double-blind and placebo-controlled study . Therefore, it is conceivable that the Syk pathway genes differentially expressed in this study could help identify patients more likely to benefit from therapy with SYK inhibitors. Additionally, Syk is critical to TNFα-induced responses , raising the possibility that the Syk pathway 47-gene signature could be used to predict increased TNFα activity prior to choosing a biologic therapeutic agent. Additionally, the increased expression of Syk pathway genes could identify patients at increased risk to develop erosive disease and could become a prognostic tool. Lastly, the Cia5a gene itself has the potential to become a new target for therapies aimed at reducing articular damage via inhibition of Syk pathway genes, including processes downstream from Syk such as NFκB.
While several genes with pro-inflammatory, proteolytic, innate immunity and inflamasome-related activity were expressed in reduced levels in DA.F344(Cia5a) congenics, groups of genes with known anti-inflammatory properties were expressed in increased levels in congenics. These genes included Timp3, Ptpn11, antagonists of reactive oxygen species (Cat, Gss, Sod1) and nuclear receptors. Nuclear receptors such as Lxra, Pparg and Rora have been shown to interfere with NFκB and AP-1 activation [39–41], and to have anti-inflammatory and arthritis-suppressive properties [42–45]. Rxrg was another nuclear receptor expressed in significantly increased levels in DA.F344(Cia5a) congenics. While Rxrg itself has not been studied in the context of arthritis, it dimerizes with Lxra, Pparg, and with Vdr, and is required for their anti-inflammatory activity. Additionally, several nuclear receptor-inducible genes, including the inflammation-suppressor Scd1 were expressed in increased levels in the synovial tissues of the congenics. These observations suggest that not only nuclear receptor levels were increased, but also their activity. We have recently identified a similar nuclear receptor expression signature in another arthritis-protective congenic strain, DA.ACI(Cia25) , suggesting that this effect is not specific to the Cia5a locus, but more broadly correlates with preservation of both a normal synovial environment and articular architecture.
The gene with the most significantly increased expression in DA compared with congenics was Tnn (Tenascin N). While little is known about this secreted extra-cellular matrix glycoprotein, it has been implicated in cancer-associated angiogenesis , and in integrin-dependent cancer motility . Another member of the tenascin family, Tenascin C (Tnc), was recently shown to be an endogenous activator of TLR4, an inducer of IL-6 and TNFα, and was required for joint damage in arthritic mice , suggesting that Tnn could have a function similar to Tnc in arthritis.
Lastly, we considered the possibility that a polymorphism in the 5’ UTR or 3’UTR region of the gene accounting for Cia5a could interfere with its transcription and/or mRNA stability, respectively, leading to increased or reduced gene-specific mRNA levels. We looked for differentially expressed genes and genes preferentially expressed by only one of the strains and located within Cia5a as a clue to the above possibility. Thirty-eight genes met these criteria, and particularly the most significant sixteen genes are interesting candidates that will be studied in detail (Table 6).