T1D develops from a breakdown in central or peripheral tolerance that is partially controlled by PTA expression in the thymus and lymph nodes, respectively. Here we demonstrate that Deaf1 plays a role in regulating PTA gene expression in peripheral lymphoid tissues. We found that the transcriptional activity of Deaf1 can be inhibited by alternatively spliced variants of Deaf1, and that the expression of these splice variants was significantly higher in the PLN of T1D patients and 12 week old NOD mice than in controls. These findings suggest that alternative splicing of Deaf1 could play a key role in the pathogenesis of T1D and NOD disease.
The age of 12 weeks is pivotal in the progression of NOD disease, as infiltrative insulitis and β-cell destruction begins at this age7
. Peripheral tolerance to certain PTAs is facilitated in the lymph nodes through the ectopic expression of PTA genes in stromal cells2–4
, and defects in peripheral tolerance may induce infiltrative insulitis. The expression of genes encoding various pancreatic PTAs is diminished in the PLN of NOD mice at 12 weeks of age5
. This coincides with a downregulation of DF1
and upregulation of DF1-VAR1
gene expression. Since DF1-VAR1 inhibits PTA gene expression and interferes with the transcriptional activity of DF1, a change in Deaf1 isoform expression may result in decreased PTA expression in 12 week old NOD PLN and reduced tolerance to these antigens.
Due to the obvious practical difficulties in obtaining human PLN tissues from pre-diabetic individuals as well as those at disease onset, we could not assess the expression of Hu-DF1 and Hu-DF1-VAR throughout all stages in the natural history of T1D. In the one pre-diabetic PLN sample we obtained, Hu-DF1-VAR expression was higher than in the PLN of all the healthy control samples examined. Interestingly, the increased expression of Hu-DF1-VAR and absence of INS in the PLN appeared to be maintained well after the onset of T1D.
Increased expression of the Deaf1 variant can inhibit the transcriptional activity of canonical Deaf1 by forming hetero-dimeric complexes that retain the canonical isoform in the cytoplasm. Both DF1-VAR1 and Hu-DF1-VAR lack the NLS domain that is required for nuclear localization and activation of gene transcription8
, and both induced minimal transcriptional activity of the 26-bp Deaf1 response element compared to that induced by DF1 and Hu-DF1. The canonical isoform of Deaf1 is structurally similar to Aire, which also contains a SAND domain and can induce PTA gene expression by interacting with modified and unmodified histone H3 via its PHD-ZF domain9, 10
. We suggest that Deaf1, rather than Aire, plays a role in controlling PTA expression in the PLN. Deaf1 may regulate PTA expression in a manner analogous to Aire since both proteins contain similar functional domains: Deaf1 contains a C-terminal ZF-MYND domain that is structurally similar to the PHD-ZF, as well as a SAND domain that functions as a DNA binding domain for chromatin-dependent transcription and a site for protein-protein interaction11, 12
. However, the effect of Deaf1 function may be cell-dependent. For example, Deaf1 represses and enhances promoter activity of the 5-HT1A receptor in presynaptic and postsynaptic neurons, respectively13
. In addition, some genes were upregulated while others were downregulated in Deaf1
-KO mice, suggesting that Deaf1 may interact with cell-specific regulatory factors to either stimulate or suppress gene transcription. The requirement for additional factors may explain why overexpression of DF1 did not significantly increase PTA gene expression in NIH 3T3 cells, and why changes in PTA gene expression in PLN tissue did not necessarily correlate with increased DF1 expression.
BALB/c mice, in which Deaf1
was knocked out, did not manifest an obvious autoimmune phenotype. However, like Aire-KO mice, the serum of Deaf1
-KO mice contained auto-antibodies that were immunoreactive against proteins in the retina of the eye1
. Interestingly, our microarray data showed that the most downregulated gene in the PLN of Deaf1
-KO mice is dopachrome tautomerase
), which encodes a protein that is expressed exclusively in the retina. In addition, three other genes among the top 30 genes that were downregulated in the PLN of Deaf1 mice (1500016O10Rik
, and Sgne1
) are expressed in the retina. The control of retinal antigen expression by Deaf1 may be of particular significance, as reduced antigen presentation in eye-draining lymph nodes prevents autoreactive T cell deletion and contributes to the development of retinal autoimmunity14
Variations in thymic PTA gene expression can influence susceptibility to autoimmune disease15, 16
. The expression of genes encoding certain PTAs was reduced in the PLN of Deaf1-
KO mice, while surprisingly, the expression the same genes and of Aire
was increased in the thymus of the Deaf1
-KO mice. This suggests the possibility that enhanced central tolerance mechanisms, mediated by increased Aire-regulated expression of PTAs in the thymus, may result in the lack of overt autoimmunity in the Deaf1
-KO mice. Strain-dependent variations in the thymic expression of genes encoding certain PTAs, such as the uveitogenic retinal antigen interphotoreceptor retinoid binding promoter (IRBP), have been observed17
. Previous studies have also shown that the severity of the autoimmune phenotype of Aire-knockout mice depends on the genetic background. Aire-deficient mice developed severe exocrine pancreatitis in NOD but not in C57BL/6 or BALB/c backgrounds18
, while autoimmune gastritis and auto-antibodies against Mucin-6 developed in Aire-deficient mice in NOD and BALB/c backgrounds, but not in the C57BL/6 background19
. The phenotype of the Deaf1
-KO is also strain-specific. In the C57BL/6 background, Deaf1
-KO mice suffered from various developmental defects that were not observed in BALB/c Deaf1
Ambp, Fgb, Ppy
were downregulated in the PLN of Deaf1
-KO mice, and we selected these genes for further study based on their regulation by Aire in the thymus1
and reduced expression in the PLN of 12 week old NOD mice. In mTECs and LNSCs, PTAs are expressed in low amounts. For example, PTA expression in lymph nodes was assessed using the iFABP-tOVA transgenic mouse model, where OVA (ovalbumin) represents a self-antigen expressed under the control of the fatty acid binding protein (FABP) promoter2
. OVA mRNA expression was detected in lymph nodes by RT-PCR, but OVA protein was not detected by immunoblotting. Similarly, PTA expression is low in the thymus, as only a small subset of mTECs ectopically express Aire and PTAs1, 21, 22
In humans, INS was not detected in the PLN of T1D patients, but was expressed in the PLN of healthy individuals and spleens of both control and T1D samples. The lack of INS expression correlated well with the high expression of Hu-DF1-VAR expressed in the PLN of T1D patients. In 12-week old NOD PLN, Ins2 gene expression was also reduced, but we could not detect a difference in Ins2 mRNA expression in the PLN of Deaf1-KO mice compared to BALB/c control mice. This may be due to the significantly lower expression of Ins2 mRNA in the PLN of Deaf1-KO and BALB/c control mice (30 to 1700-fold lower) compared to that of NOD and NOD.B10 mice.
Members of the Reg family have been described as autoantigens in T1D, and the inflammation-induced upregulation of Reg
expression in islets was suggested to contribute to the premature onset of diabetes23
. We showed that Reg3g
expression was downregulated in the PLN of Deaf1
-KO mice and 12-week old NOD mice, but were unable to determine if Deaf1 directly regulated Reg3g
expression since the NIH 3T3 and LN CD45-
cell lines that we used for the siRNA experiments did not express measureable amounts of Reg3g
. A loss of endogenous PTA gene expression has been shown to occur in cultured mTEC24
and LNSC lines (unpublished data). Thus, future experiments involving monoclonal antibodies to DF1-VAR1 and/or in situ hybridization studies of primary lymph node stromal elements derived from the PLN of NOD and NOD.B10 mice may be necessary to identify the actual stromal cell that expresses the variant Deaf1 isoforms and to determine the direct role of Deaf1 in the expression of PTA genes.
While the expression of Ambp, Fgb, Ppy, and Reg3g is regulated by Aire in the thymus, the expression of these genes in the PLN is more likely regulated by Deaf1. Aire expression was not altered in the PLN of Deaf1-KO mice or NOD mice, and most of the PTAs regulated by Aire in eTACs appear to be distinct from those regulated by Deaf1 in the PLN.
Our study suggests that Deaf1 promotes the ectopic expression of genes encoding PTAs in the PLN, and PTA expression in LNSCs can mediate peripheral tolerance2
. Thus, fine-tuning of peripheral tolerance, at least in the PLN, may occur through alternative splicing of Deaf1
. Alternative splicing is a mechanism that is often used to control immune responses. However, variations in splicing can impair immune function and contribute to various autoimmune diseases25
. It is unclear how splicing of Deaf1
is controlled, but inflammation of the NOD PLN may be involved since various inflammatory cytokines have been shown to induce alternative splicing26–30
. Differences in Deaf1
splicing in NOD vs. NOD.B10 mice may also be due to other genes within the Idd1
susceptibility region, a MHC congenic interval that distinguishes the two strains. Allelic variation of such genes or differences in their regulatory regions may influence their expression or function, which may be associated with splicing events.
We propose that during the progression of T1D and NOD disease, alternative splicing of Deaf1 occurs in the PLN. The alternatively spliced Deaf1 variant interacts with and retains the canonical isoform in the cytoplasm and inhibits PTA gene expression. Decreased expression of pancreatic antigens in the PLN may impair peripheral tolerance and lead to the survival of autoreactive T cells specific for these antigens. Thus, we suggest that differences in the expression of Deaf1 isoforms in the PLN of humans with T1D and NOD mice may contribute to the development of this disease.