In the present study, we generated Tank−/−
mice and showed that TANK is essential for negative regulation of canonical NF-κB signaling. Tank−/−
mice displayed enhanced activation of macrophages and B cells in response to TLR ligands and antigens, culminating in the development of fatal immune complex-mediated renal failure. Although TANK has been shown to positively regulate TBK1 and IKK-i
-mediated type I IFN production by in vitro
studies, analyses of Tank−/−
mice revealed that TANK was not needed for activation of the type I IFN pathway downstream of RLRs or TRIF. TANK forms a family with NAK-associated protein 1 (NAP1) and similar to NAP1 TBK1 adaptor (SINTBAD)38, 39
, which are comprised of an N-terminal coiled-coil domain and a TBK1-binding domain. NAP1 and SINTBAD have also been implicated in the activation of TBK1 and IKK-i
downstream of virus sensors. Knockdown of NAP1, SINTBAD or TANK by siRNA has been linked to impaired IFN responses. Hence, it is possible that these three proteins function redundantly in the activation of TBK1 and IKK-i
Although previous studies showed that TANK is a positive regulator of NF-κB, our results clearly demonstrate that TANK is critical for the negative regulation of canonical NF-κB via suppression of TRAF6 ubiquitination. K63-type ubiquitination is important for the activation of TAK1 via TAB2 and TAB3 in TLR signaling, and TANK may inhibit TRAF6 ubiquitination by directly binding to TRAF6 in response to TLR stimulation. Although A20 and CYLD have been identified as deubiquitinases40–42
, TANK does not harbor a deubiquitination enzyme domain. Immunoprecipitation experiments revealed that overexpressed A20 or CYLD failed to co-immunoprecipitate with overexpressed TANK, suggesting that TANK may suppress ubiquitination of TRAF6 independently of A20 or CYLD (data not shown). Further studies are required to assess the precise mechanism through which TANK modifies TRAF6. In addition, canonical NF-κB activation in response to BCR and CD40 stimulation was augmented in Tank−/−
B cells. Consistently, proliferation of B cells in response to TLR and BCR stimulation was highly elevated in Tank−/−
mice. In TCR signaling, TRAF2 and TRAF6 were reported to participate in NF-κB activation downstream of Bcl10 and MALT143
. Given that TANK suppresses the polyubiquitination of TRAF6 in response to TLR stimulation in macrophages, it is possible that TANK suppresses BCR and CD40 signaling by regulating the activation of TRAF proteins in B cells. On the other hand, activation of non-canonical NF-κB activation was not enhanced in Tank−/−
B cells, and it was reported that TRAF3 mainly controls non-canonical NF-κB activation in B cells44
. Hence, these observations suggest that TANK is not involved in signaling downstream of TRAF3. Further, TRAF2 can control non-canonical NF-κB as well as marginal zone B cell development. The relationship between TANK and TRAF2 needs to be further explored in future.
The disease caused by the absence of TANK was characterized by glomerulonephritis due to deposition of immune complexes in the glomeruli. In addition, anti-dsDNA Abs and ANA were present in high concentrations in Tank−/−
mice. These observations indicate that Tank−/−
mice may represent a mouse model of lupus-like immune diseases. The phenotypes of Tank−/−
mice are reminiscent of mice overexpressing IL-6 in B cells45
, which are characterized by lymphadenopathy and plasmacytosis culminating in the development of severe glomerular nephritis. IL-6 is a pleiotropic cytokine responsible for fever, acute-phase protein expression, osteoclast activation and the development of TH
-17 and plasma cells. Indeed, Tank−/−
macrophages showed enhanced production of proinflammatory cytokines including IL-6 and TNF in response to TLR stimulation. Furthermore, Tank−/−
mice failed to produce autoantibodies and did not develop glomerulonephritis in the absence of IL-6. These results indicate that IL-6 is essential for the development of the Tank−/−
B cells that are responsible for the production of autoantibodies. In contrast, Tank−/−
T cells responded normally to TCR stimulation. Given that TANK is critical for inhibiting BCR-induced B cell activation, it is possible that the lack of TANK in B cells is important for the generation of autoimmune nephritis via aberrant activation of B cells in response to antigen stimulation.
The generation of anti-dsDNA Abs in Tank−/−
mice was significantly decreased in response to oral treatment with antibiotics or in the absence of MyD88, suggesting that TLR signaling is critical for the development of autoimmune diseases in Tank−/−
mice. Although various proteins have been identified as negative regulators of TLR signaling, few mice lacking any single one of these proteins spontaneously develop autoimmune diseases spontaneously, with the exception of mice lacking A20. A20−/−
mice spontaneously develop multiorgan inflammation and premature lethality, which can be rescued by MyD88 deficiency46, 47
. Unlike Tank−/−
mice do not develop immune complex-mediated glomerulonephritis. A20 controls TNFR in addition to TLR signaling, and the responses to TNF were not altered in Tank−/−
cells. TNF is involved in the pathogenesis of organ-specific autoimmune diseases, such as rheumatoid arthritis and Crohn’s disease48
. Hence, the differences in the signaling pathways regulated by A20 and TANK may explain the differences in the types of autoimmune disease caused by A20 or TANK deficiency.
Since oral treatment with antibiotics ameliorated autoantibody production in Tank−/−
mice, constitutive stimulation of TLRs by intestinal microflora seems to be responsible for the generation of autoimmunity in the absence of TANK. Bone marrow transfer experiments revealed that hematopoietic cells were responsible for the lethality in Tank−/−
mice (data not shown). Intestinal microflora contribute to the pathogenesis of IBD48, 49
, and the colitis observed in IL-10-deficient mice was rescued by the absence of MyD8824
, suggesting that TLR signaling is involved in the pathogenesis of IBD. Since TLRs are expressed on intestinal DCs and are responsible for sensing microbes in the intestine, it is possible that TANK controls the production of certain cytokines in intestinal tissues. Further studies are required to understand why TANK deficiency causes autoimmune nephritis but not colitis.
In addition, the antigen-specific humoral immune responses to haptens were enhanced in Tank−/− mice. This may be due to the enhanced DC and B cell activation in response to antigens and the adjuvant in Tank−/− mice. It will be interesting to explore whether inhibition of TANK expression in certain cell types is beneficial for inducing antigen-specific immune responses in vivo. Modification of TANK may be useful in vaccinations when administered together with an adjuvant.
In summary, the results of the present study clearly demonstrate that TANK is a negative regulator of TLR and BCR responses. Future studies involving cell-type specific deletion of TANK will clarify the complex interplay between immune cells needed to prevent the development of autoimmune diseases.