CX3CL1/CX3CR1 signaling has been established as an important pro-inflammatory chemokine receptor signaling interaction in chronic inflammatory diseases (reviewed in (5
)) including RA (1
) and atherosclerosis (10
). Correlation studies have shown increased expression of CX3CL1 and/or CX3CR1 in RA (34
) and that they are more elevated in patients with severe disease (35
). In a mouse CIA model, treatment of arthritic mice with a neutralizing antibody to the unique ligand for CX3CR1, CX3CL1, showed protection from disease that was mediated by inhibition of macrophage/monocytes trafficking to the joint (8
). However, beyond impairment in macrophage/monocyte trafficking, changes in B and T cell function were not observed in these studies (8
CX3CL1 is highly expressed on endothelial cells, intestinal epithelial cells, synoviocyte-like fibroblasts, and on some dendritic cells (6
). In contrast, its receptor CX3CR1 is expressed mostly on immune cells such as monocytes, macrophages, dendritic cells, NK cells, a subset of T cells, and recent reports that suggest a subset of B cells (11
). CX3CL1/CX3CR1 signaling activates the proinflammatory NF-kB pathway (39
), and CX3CR1 deficiency is associated with decreased IL-6 and TNF-α production by macrophages and dendritic cells (40
). Because of its predominant expression on immune cells and multiple implicated mechanisms of immune regulation, we examined the genetic deletion of CX3CR1 in the CIA model to determine whether or not additional mechanisms beyond that of cellular trafficking may be affected in inflammatory arthritis when the receptor, as opposed to the ligand, was targeted.
Our studies show that CX3CR1 deficiency confers protection in CIA, in part, through decreased humoral and T cell responses. Since professional antigen presenting cells (i.e. monocytes, macrophages, B cells, and dendritic cells) have CX3CR1 on their cell surface (9
), it is conceivable that alteration of the CX3CR1+ subset of these antigen-presenting cells could lead to decreased production of autoantibodies, which are known to be pathogenic (20
). Recently, Corcione et al. immunized CX3CR1-/- and wildtype mice with ovalbumin (OVA) to determine whether or not CX3CR1 deficiency had effects on antigen-specific antibody formation. Their study had findings similar to ours in that OVA-specific IgG production was decreased (26
). Lymphoid follicle architecture, B cell, and T cell enumeration did not differ, thus the authors concluded that differences in autoantibodies could not be directly determined. Interestingly, a study by Bar-On et al (27
) has identified a specific subpopulation of CX3CR1+ CD8α+ dendritic cells that share a gene signature overlapping with plasmacytoid dendritic cells. Since plasmacytoid dendritic cells are known for regulating B cell differentiation and antibody production (41
), this CX3CR1+ dendritic cell subset may have functions that affect antibody production in inflammation.
The Th17 subset is recently recognized as an important pro-inflammatory mediator in RA (30
). Neiss et al. showed that CX3CR1+ peptide-pulsed dendritic cells preferentially supported the differentiation of CD4+ Th17 cells in vitro
in an inflammatory bowel disease model (40
). Our study did not see a difference in the ability of CX3CR1-/- mice to develop Th17 skewing in vitro
, albeit there was a trend toward decreased Th1 IFN-γ production, which would support the conclusions of Neiss et al. We also acknowledge that only a small subset of CD4+ T cells express CX3CR1 () after immunization for CIA, which may have limited our ability to detect a difference in the cytokine production assays.
An additional or alternative mechanism beyond that of impaired Th17 induction in CX3CR1-/- lymph node cells could be the selective impairment of this particular T-helper subset to migrate to the joint. Although we were able to detect a subpopulation of CD4+ T cells that also expressed CX3CR1 above background levels (), the total numbers of IL-17+ CD4+ CX3CR1+ cells are low enough (5% IL-17+ of the 1% CD4+ CX3CR1+ lymphocytes) that this raises questions as to the accuracy and reproducibility of this measurement. The ideal experiment would be to examine CIA responses and CD4+ IL-17+ cells using the CX3CR1GFP
reporter mice (4
), which have green fluorescent protein knocked into the CX3CR1 gene locus. In this way, a more sensitive and accurate examination of the CX3CR1 expression pattern and migration of small cellular subsets that normally express this receptor (such as Th17 cells) could be more reliably ascertained and studied. Although direct migration and enumeration of CD4+ IL-17+ CX3CR1+ cells in CIA was limited, we were able to show 3-fold decreased numbers of CD4+ IL-17+ cells in the inflamed paws of CX3CR1-/- mice (). Although altered migration is suggested in that there are fewer Th17 cells in the CX3CR1-/- inflamed paw, the evidence is indirect, and we cannot exclude the possibility of other mechanisms such as enhanced apoptosis or impaired proliferation of Th17 cells as well.
Based on our findings of decreased IL-23 in the joint, we additionally postulate that within the joint cytokine milieu, there may be environmental differences that affect local T cell cytokine secretion and function. Specifically, IL-23 is secreted by dendritic cells and induces the production of IL-17 by T cells (42
). IL-23 is not needed for the de novo
generation of Th17 cells but can augment IL-17 production from already generated Th17 memory cells (43
). Consequently, decreased IL-23 within the local microenvironment of the joint may additionally affect the function of Th17 cells in CX3CR1-/- mice.
The CIA model is an approximation of human RA and does have limitations. Particularly, the inflammatory reaction is robust, and even in modest disease, extensive damage to the joint is seen at early and chronic time points. Therefore, a reduction, as opposed to elimination of disease, may not achieve statistical significance in histopathology scores in this model. One of our proposed mechanisms of protection is that loss of CX3CR1 impairs cellular trafficking to the joint, particularly of cells involved in the Th17 axis. However, inflammatory cells are not completely inhibited in trafficking to the joint, as seen on the histopathology sections (), which may explain why disease is not significantly different at later time points. Additionally, matrix metalloproteinases (MMPs), which implement much of direct tissue destruction in CIA, are not different between CX3CR1-/- and wildtype mice, which could explain why bony erosions and cartilage loss are not significantly different between groups.
Our group has previously published on the importance of CX3CL1/CX3CR1 signaling in atherosclerosis, which is attributed in part to alterations in inflammatory monocyte and dendritic cell trafficking to affected lesions (9
). Accelerated atherosclerotic disease from longstanding RA is becoming a widely recognized long-term complication (13
). In a recent observational study by Pingiotti et al., peripheral blood isolated CD4+ CX3CR1+ T cells from RA patients were expanded when compared to healthy controls (34
). Further, this increase in CD4+ CX3CR1+ T cells from the RA patients correlated with increased carotid intima-media thickness (IMT) and the Disease Activity Scoring system (DAS 28) in Rheumatoid Arthritis (34
). These data suggest that CX3CL1/CX3CR1 blockade may have long-term benefits that extend beyond inflammatory arthritis and into prevention of early endothelial dysfunction leading to atherosclerotic disease in these patients.