Alterations in the self-renewal and differentiation ability of bone marrow mesenchymal stem cells in a mouse model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic disruptive polyarthritis. There is no cure for RA and today's treatments aim at achieving the lowest possible level of arthritis disease activity. In the UK alone RA costs the health system around £560 million a year, and £1.8 billion a year in absence from work. The events preceding the disease and leading to its initiation and progression are unknown. Thus, an understanding of these processes is crucial for the identification of new and more cost-effective treatments and for the move from a manageable to a curable disease.

Although the main disease site is the synovium, there is growing evidence that the bone marrow (BM) is actively involved and may even be the primary initiating site of the disease [1]. Abnormalities in both the haemopoietic progenitor cells and the BM stroma have been described [2]. Patients with active RA have been seen to exhibit low frequency and accelerated apoptosis of BM CD34+ cells and defective clonogenic potential [2]. Difficulties in haemopoietic stem cell (HSC) transplantation with very common, but unfortunately, relatively short-lived responses have been reported [3]. Moreover, age-inappropriate shortening of telomeres in CD4 T cells and granulocytes have been shown not only in patients affected by RA but also in healthy adult HLA-DR4 donors, the HLA haplotype with the major genetic risk factor for RA [4]. These data suggest that HSC undergo increased replicative stress not only in patients affected by RA but also in patients predisposed to RA and it can occur independently of the rheumatoid disease process. The causes of the increased apoptosis and replicative stress of HSC in patients affected by RA are unknown.

HSC requires a balanced supportive environment, the niche, to survive, proliferate and appropriately differentiate. An impaired hematopoiesis-supporting capacity of BM stroma has been described in RA patients [2]. However, it is unknown what the changes are in the bone marrow (BM) niche, which may lead to ineffective support of HSC. Mesenchymal stem cells (MSCs), and their differentiated progenies, including osteoblasts and adipocytes, have been shown to be important elements of the BM niche and have a role in HSC survival, proliferation and differentiation [5‐8]. Cells with properties of MSC have been found to increase in the synovium of patients affected by RA and are thought to be one of the main causes of perpetuation of the disease, suggesting a possible dysregulation of MSC proliferative capacity [9]. In this study, we hypothesize that bone marrow MSCs undergo enhanced self-renewal at the onset of RA-like disease.

To test this hypothesis we have used an Interleukin-1 receptor antagonist knock out mouse model of RA on a Balb/c background (Balb/c IL1ra-/-), which spontaneously develops inflammatory arthropathy with inflammatory cellular infiltration, pannus formation and periarticular bone erosions, similar to RA in humans, with 100% penetrance by weeks 20 of age [10]. We have determined the changes present in the bone marrow microenvironment before disease onset and with development of RA-like disease.

This study showed the presence of important changes occurring in the bone marrow niche before disease onset in relation to mesenchymal progenitors, adipogenesis and osteoclastogenesis. It identifies progressive amplification of mesenchymal progenitors in the bone marrow with disease onset and progression but impairment of their differentiation to the osteogenic lineage.

The role of the bone marrow niche and mesenchymal stem cell in haemopoiesis and regulation of inflammation is becoming progressively recognised [14‐16]. Despite the fact that in RA there are clearly defined disturbances in the haemopoietic and immune system, changes in bone marrow mesenchymal stem cells and their progeny, which may drive those modifications, have not been studied. To address this question we have used the IL1ra-/- on a Balb/c background. This model has histopathological features, which closely resemble arthritis present in patients affected by RA with synovitis, inflammatory cellular infiltration, which includes lymphocytes, pannus formation and periarticular bone erosions. The knocking out of the interleukin-1 receptor antagonist is permissive but not solely responsible for the occurrence of RA-like arthropathy as the occurrence depends also on the mouse strain. Moreover, the histopathological features of the disease differs from those described in patients who harbour a deletion of the interleukin-1 receptor antagonist gene recently described [17]. In those patients there is multiple chronic osteomyelitis, and bone erosions are intraosseous and not periarticular as seen in our murine model or in patients affected by RA. Therefore, the Balb/c IL1ra-/- is a valuable model to study events which occur over time and in the BM. Obtaining BM samples from patients affected by RA and who have not undergone treatment is difficult.

Here, for the first time, we provide evidence of changes in the composition of the bone marrow niche present already before the development of RA-like disease. We have shown a deficiency in mesenchymal progenitors with adipogenic potential and bone marrow adiposity together with a decrease in the maturation of osteoclasts before disease development. Moreover, this was present only in Balb/c IL1 ra-/- mice, who progressed to develop RA-like disease and not in C57BL6 IL1ra-/-, which have been shown not to develop RA-like arthritis, suggesting an association of these abnormalities with RA development. Although it is difficult to predict the precise cascade of events leading from low adipogenesis to decreased activation of osteoclasts, there is evidence that the two processes may be linked. Mature adipocytes release adipokines such as leptin, ghrelin and adiponectin, or growth factors such as monocytes chemotactic protein 1 (MCP-1) [18]. For examples, MCP-1 has been shown to mediate osteoclastogenesis and bone resorption [19]. Further studies are now needed to understand the genetic alterations and the cascade of events leading to those abnormalities.

Regardless of the precise cascade of events, our data are in agreement with other published data. In the study by Naivares et al (2009) low bone marrow adiposity has been positively correlated with the ability to support proliferation of hematopoietic progenitors [8]. Lack of adipogenesis in AZIP/F1 recipient mice (genetically incapable of forming adipocytes) enhanced hematopoietic recovery after lethal irradiation by enhancing engraftment of short term progenitors [8]. Adipocyte-rich marrow was shown to harbour a decreased frequency of progenitors and relatively quiescent stem cells [8]. Therefore the reduction in adipogenesis in Balb/c IL1ra-/- mice supports the notion of an enhanced stimulation of the HSC to proliferate and is in agreement with the study by Schonland et al. (2003) where individuals with the HLA-DRB104 haplotype, shown to be more at risk for developing rheumatoid arthritis, had accelerated telomere shortening of both lymphocytes and neutrophils, a marker of cellular proliferation and ageing of haemopoietic progenitors [4].

Dysregulated adipogenesis may be responsible for the establishment of a proinflammatory environment. Adiponectin has been suggested to have a role in vivo for immediate moderation of inflammatory reactions occurring after invasion by foreign pathogens and prevents an excessive and prolonged inflammatory response. Indeed lipopolysaccharide (LPS) induced expression of TNFα mRNA in macrophages and was markedly suppressed by pre-treatment with recombinant adiponectin [20]. Adiponectin pre-treated macrophages failed to release TNF-α in response to LPS [20]. Therefore it is not unreasonable to suggest that the low number of adipocytes found in the bone marrow of Balb/c IL1ra-/- mice may lead to reduced levels of adipokines, such as adiponectin, which in turn reduce activation of macrophages and allows increased presence of inflammatory cytokines such as TNF-α, following the start of any inflammatory process. Indeed, elevated levels of TNFRII, a surrogate soluble receptor, which parallel levels of TNF-α, has been found elevated in the preclinical phase of disease of patients that go on to develop RA up to eight years after [21].

MSC share many properties with fibroblast-like synoviocytes (FLS) [22], the cells responsible for the thickening of the synovium in the joint in RA and perpetuation of the inflammatory process by recruiting and supporting the survival of inflammatory cells [23]. It is thought that MSC may even contribute to the FLS pool in the joint. When the bone marrow of green fluorescence protein (GFP) transgenic donor mice was transplanted into lethally irradiated recipient mice, it was shown that normal FLS contained a minor fraction (1.2%) of GFP+ cells [24]. As FLS undergo increased proliferation it is not unreasonable to suggest that MSC may undergo expansion in the bone marrow too at disease onset and change the composition of the bone marrow niche. Our data point to increased numbers of MSC following establishment of the disease. Our findings are not in agreement with a previous study measuring the number of bone marrow mesenchymal progenitors in patients affected by RA, where no significant difference was found [25]. The reason for the apparent discrepancy may lie in the fact that most patients included in the study by Kastrinaki et al. (2008) [25] had undergone treatment and those who had not undergone treatment were a very small number with a wide age range. This may have compromised the detection of any difference, especially considering that the number of CFU-F tend to decrease with age [12].

Of interest is the accumulation of progenitors, which are unable to differentiate to the osteoblast lineage. The mechanism leading to MSC accumulation and blocking of their differentiation are unclear and likely due to a complex interplay of cytokines and cellular cross talks, mostly related to the in vivo inflammatory environment. When these cells were induced to differentiate to the osteoblast lineage in vitro they showed formation of colony forming unit-osteoblasts, which were alkaline phosphatase positive and did not differ in the pattern of staining from those derived from the bone marrow of their wild type age-matched controls. Proinflammatory cytokines such as TNF-α and IL1β have been shown to block osteogenic differentiation in vitro [26, 27] and support our findings. The accumulation of MSC, known to secrete RANKL, may also lend an explanation to the loss of defective osteoclastogenesis observed in the Balb/c IL1ra-/- animals before disease onset. Indeed, our data shows that with the establishment of disease the number of osteoclasts is no longer significantly decreased and, if anything, it exhibits a trend to increase. This is in agreement with the well-known increase in the number of activated osteoclasts reported in the joints of patients with RA and with data suggesting that at very early stages of inflammation, when levels of TNF-α are low, mesenchymal stem cells are required for osteoclastogenesis [28]. All together our data are in agreement with reports of RA patients developing systemic osteoporosis with time [29] and of systemic accelerated bone loss in another model of inflammatory arthritis mainly as the result of arrested osteoblast function [30]. Ways to induce effective differentiation of the mesenchymal progenitors to the osteoblast lineage may provide both a way to reduce expansion of MSC and prevent systemic bone loss.

The accumulation of MSC raises the intriguing question whether these cells, known for their immunosuppressive properties, are exerting the expected anti-inflammatory action and whether the proposed administration of additional MSC, as novel therapeutic strategy in RA, would have the desired effect of dampening the auto-immune responses. Despite a wealth of literature describing the immunosuppressive ability of MSC, more recently a pro-inflammatory phenotype of MSC has also been described [31, 32]. This requires activation of TLR4 and possibly TLR3 receptors present on MSC. Indeed overexpression of TLR3 and TLR4 has been described in synovial fibroblasts of patients affected by RA and beg the question whether bone marrow mesenchymal stem cells in RA patients show similar overexpression and have a pro-inflammatory phenotype as a result of their engagement [33]. The fact that administration of murine MSC in models of RA disease has given contrasting results [34, 35] highlights that this is a potential problem and calls for a closer analysis of the inflammatory phenotype of the patients' MSC at the time of active disease, following expansion before administration and after administration to the patient. This may help to determine whether MSC preparation varies in terms of predisposition to acquire a pro-inflammatory phenotype or whether this is an acquired characteristic as a result of contact with the patients' cellular environment. Moreover, it may establish whether the cellular environment confers the pro-inflammatory phenotype depending on the disease stage and status of remission.

We have shown an accumulation of MSC progenitors with the onset of RA-like disease and a block in their differentiation to osteoblasts. These data highlight the need for therapeutic intervention with molecules capable of inducing differentiation of MSC to osteoblasts to prevent the onset of osteoporosis. Moreover, it puts into question whether additional administration of MSC proposed as treatment to reduce the autoimmune response [30] is required and would be efficacious. More studies into the molecular mechanism leading to enhanced maintenance of MSC in RA-like disease, their pro-inflammatory phenotype and ways to interferes with this are required. Small molecules such as glycogen synthase kinase -3 inhibitors have been shown to have anti-inflammatory properties and to drive commitment of MSC to osteogenic lineage [36, 37], suggesting they may have a therapeutic impact in both reducing inflammation and bone loss.