In this manuscript we used a mouse model of Graves’ disease (EAGD) to test the hypothesis that thyroidal over-expression of CD40, driven by a CD40 Kozak SNP, plays a role in the etiology of Graves’ disease. However, in contrast to humans, where it is well documented that CD40 is expressed and functional on thyroid follicular cells, both in normal and Graves’ glands (16
), we found that wild type mice express very low levels of CD40 in the thyroid. Therefore, to test this hypothesis, we created a transgenic mouse over-expressing CD40 in the thyroid (Supplemental Figure 1
). Our data demonstrated that thyroid specific over-expression of CD40 plays a role in the pathogenesis of Graves’ disease. TG mice had significantly higher levels of TSHR stimulating antibodies resulting in more severe thyrotoxicosis. This was a specific effect and not the result of global increase in immunoglobulins, since the levels of total immunoglobulins and their isotypes were not different between TG and WT mice (Supplemental Figure 4
Mechanistically, in thyroids from EAGD mice IL-6 expression was found to be augmented in the setting of over-expression of CD40 in the thyroid, suggesting a role for IL-6 secretion, triggered by thyroidal CD40 stimulation, in the production of tissue specific antibodies. Indeed, IL-6 has been shown to play an important role in adaptive immunity, specifically in inducing B-cell differentiation into antibody producing plasma cells (31
). To confirm the role of IL-6 in antibody production in EAGD, we blocked IL-6 in TG and WT EAGD mice, and analyzed the production of thyroid specific antibodies. While there was no difference in TRAb and T4 levels in WT mice treated with anti-IL-6 antibody, this treatment delayed production of TRAb in TG mice. Moreover, it appears that blocking IL-6 restricted the production of pathogenic (stimulating) TRAb, since T4 levels in anti-IL-6 treated CD40-TG mice remained within the normal range, even though the titers of antibodies eventually returned to the levels of isotype control treated mice. Since this effect was seen only in the TG mice, it suggests that IL-6 plays a role in augmenting pathogenic TRAb production in EAGD in the setting of high levels of CD40 in the thyroid. Supporting this notion are the data showing that stimulation of CD40 on primary human thyroid cells resulted in the secretion of pro-inflammatory cytokines including IL-6.
The data presented here help to provide a novel mechanism for the association of CD40 with GD as well as other autoimmune diseases. Previously, we and others (9
) have shown an association of a single nucleotide polymorphism (SNP) in the Kozak sequence of the CD40 gene with Graves’ disease. The association was significantly stronger in a subset of Graves’ patients having high titers of thyroid specific antibodies, suggesting that CD40 played a role in the production of antibodies that mediate Graves’ disease (9
). Functionally, the CC genotype (associated with disease) has been shown to cause increased CD40 protein expression (10
). However it was not clear whether increased CD40 expression on B-cells, thyroid cells, or both, conferred susceptibility to GD. Here we show that thyroidal expression of CD40 plays an important role in disease etiology as over-expression of CD40 in the thyroid augmented disease and deletion of CD40 in non BM derived cells including thyroid cells attenuated disease. Since our chimeric-KO mice had CD40 deletion in all non-BM derived cells and not only in thyroid cells, it is possible that CD40 expression in other non BM cells in addition to thyroid cells may also contribute to disease etiology. Moreover, EAGD itself caused increased thyroidal expression of CD40 in WT mice which can additionally help perpetuate the disease (Supplemental Figure 3F
Our findings showing a role of target tissue expression of CD40 in the etiology of GD may be relevant to other autoimmune diseases. Indeed, in addition to being expressed in the human thyroid (16
), CD40 has been shown to be expressed in many tissues that are targets for other organ specific autoimmune conditions such as β-cells (18
), the target in T1D, spinal cord (37
), tissue affected by MS, keratinocytes (20
), cells affected in psoriasis, colon fibroblast and intestinal epithelial cells (21
), cells effected by inflammatory bowel disease (IBD), and synovial cells from RA patients (22
). Moreover, genetic associations with the CD40 gene locus have been reported in conditions besides Graves’ disease, including rheumatoid arthritis, multiple sclerosis, and asthma, suggesting that tissue specific CD40 expression may also play a role in these autoimmune diseases. In multiple sclerosis, CD40 expression is increased in the spinal cord during acute relapses of disease and deleting CD40 in the CNS compartment in experimental autoimmune encephalomyelitis has been shown to result in a less severe disease (39
), similar to our observation in mice lacking CD40 in the thyroid.
One of the major effects of CD40 stimulation is induction of cytokine secretion. Cytokine secretion after CD40 stimulation has been previously studied in target tissues of other autoimmune conditions. In the β-cell, CD40 stimulation led to the secretion of IL-6, IL-8, MCP-1, and MIP-1β (41
). On keratinocytes, stimulation of CD40 with sCD154 and IFN-γ resulted in the up-regulation of cellular adhesion molecules, the anti-apoptotic gene Bcl-x, IL-8, CCL20, RANTES, and MCP-1 (20
). In microglia cells, stimulation of CD40 caused secretion of IL-12 and TNF-α (42
). Colon fibroblasts stimulated with anti-CD40 antibody showed activation of NF-kB and production of IL-6, MCP-1, and IL-8 (21
), and intestinal epithelial cells stimulated with CD40L secreted IL-8 (38
). Finally, in synovial fibroblasts, CD40 stimulation led to proliferation, as well as increased levels of adhesion molecules and IL-6, GM-CSF, MIP-1α (43
), and RANKL (44). Thus, our data, demonstrating increased IL-6, TNF-α, and IL-8 in thyrocytes stimulated with CD40 antibody, is consistent with data in other autoimmune conditions and suggests a generalized mechanism by which CD40 tissue expression plays a role in autoimmunity. For this reason, it seems plausible that blocking the interaction of CD40 with its ligand, CD154, might suppress organ specific autoimmunity. However, the first clinical trial using anti-CD154 mAb to block the interaction of CD40-CD154 had to be discontinued early due to thrombosis in some patients (45
). Therefore, in the future, it may be more effective to target downstream effectors in this pathway. Indeed, our data suggest that blocking IL-6 may be an attractive approach to treating autoimmune diseases influenced by CD40 target tissue expression.
In conclusion, we have shown, for the first time, a novel mechanism by which CD40 expression in the thyroid may contribute to the etiology of Graves’ disease. We found that thyroidal CD40 over-expression augments the production of thyroid specific antibodies, resulting in a more severe disease, whereas deletion of thyroidal CD40 had the opposite effect. Therefore, a model is emerging whereby, during times of local inflammation (e.g. induced by infection, or other toxins such as excess iodine), thyroidal CD40 activation can result in local cytokine secretion, bystander activation of resident T- and B-cells, and increased B-cell tissue specific responses, leading to thyroid specific antibody production. When other predisposing factors are present, either environmental or genetic, this autoimmune reaction may result in the onset of clinical GD (). Therefore, our data suggest that CD40 and its downstream cytokine response may be potential therapeutic targets in Graves’ disease. Moreover, these same targets could be important in other autoimmune conditions, where target-tissue CD40 has also been shown to play an important role.
Proposed mechanism of the function of CD40 in the pathogenesis of Graves’ disease