Cutting Edge: K63-Linked Polyubiquitination of NEMO Modulates TLR Signaling and Inflammation In Vivo

Abstract

AgRAg receptor To address this issue, we introduced a point mutation (Lys 392 →Arg 392 or K392R) into the mouse germline that specifically disrupts K63-linked ubiquitination of NEMO at Lys 392 while sparing other Ubc13 substrates (NEMO-KR mice). NEMO-KR mice develop normal numbers of lymphocytes with no apparent defect in AgR signaling. However, primary macrophages and dendritic cells (DCs) from NEMO-KR mice exhibit blunted cytokine responses to TLR agonists. Consistent with this, survival rates among NEMO-KR mice are improved relative to those of control animals when challenged with doses of LPS that elicit lethal endotoxic shock. Surprisingly, NF-κB and MAPK signaling are largely unaffected in NEMO-KR cells, suggesting that Ubc13 impinges on these pathways via a mechanism independent of NEMO ubiquitination. We conclude that K63-linked ubiquitination of NEMO at Lys-392 functions as a rheostat for TLR-dependent responses in innate immunity. DC Materials and Methods NEMO-KR miceThe NEMO-targeting vector contained a point mutation in the Lys 392 codon (AAG) that converted it to an Arg codon (CGG). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (Neo) cassette was inserted into a StuI site between exons 9 and 10. The short and long homology arms were generated from the NcoI-StuI fragment encompassing exon 9 (1.6 kb) and the StuI-KpnI fragment encompassing exon 10 (4.0 kb). Generation of NEMO-KR mice (Mouse Genome Informatics no.3775942; Ikbkg tm1dwb ) was performed as described (9). All experiments were conducted according to guidelines of the Animal Care and Use Committee at Vanderbilt University (Nashville, TN). ImmunizationsKeyhole limpet hemocyanin (KLH; Sigma-Aldrich) in CFA (Difco) was injected i.p. (100 μg/ 50 μl/mouse). Serum collection and ELISA for KLH-specific Abs were performed as described (10). Cell purificationSplenic B and T lymphocytes were purified using CD43-negative selection and pan-T cell isolation kits, respectively (Miltenyi Biotec). For macrophages, mice were injected i.p. with 3% thioglycolate (2 ml; Sigma-Aldrich). Four days postinjection, peritoneal exudate cells were collected and cultured overnight in complete RPMI 1640 medium. Adherent cells were used as peritoneal macrophages. Bone marrow DC (BMDC) and macrophages were generated as described (11,12). Cell-based assaysFor cytokine responses, macrophages or BMDC were treated with agonists for 24h(5 × 10 4 cells/well). Levels of TNF, IFN-γ, and IL-6 in supernatants were measured using a BD CBA6 kit (BD Biosciences). Mouse IL-12/p40 was detected by ELISA (BD OptEIA Set) following cellular stimulation with LPS (100 ng/ml) and IFN-γ (30 ng/ml). Biochemical analysesMacrophage lysates were prepared and probed on Western blots for NF-κB and MAPK signaling components as described (8). Results Generation of NEMO-KR miceTo assess the physiologic role of NEMO ubiquitination, we introduced a point mutation into the mouse germline that prevents Ub attachment to Lys 392 . The X-linked NEMO allele in embryonic stem (ES) cel...

HSV ICP0 recruits USP7 to modulate TLR-mediated innate response

“…61,62 Macrophages and DCs from mice with germ line mutation expressing a ubiquitination-defective form of IKK␥ (K392R) had blunted TLR cytokine response and were resistant to LPS-induced endotoxic shock. 56 Here, we present evidence that ICP0:USP7 complex protects HSV against host innate immunity by directing USP7 to disassemble K63 polyubiquitin chains off TRAF6 and IKK␥. Within the TLR signaling pathway, the autoubiquitination site for TRAF6 has been mapped to lysine 124, 55 whereas IKK␥ is ubiquitinated at lysine 285 and 399 (392 in mice).…”

“…TLR-mediated cytokine response, on the other hand, requires NF-B and MAPK activity and both are dependent on K63-ubiquitin modification of signaling adaptors including TRAF6 and IKK␥. [53][54][55][56] This differential requirement for K63-ubiquitination of adaptor proteins by nonoverlapping signaling pathways allows HSV to fine-tune NF-B activity in infected cells, generating sufficient NF-B response for its own replication yet suppressing host innate response.…”

Signaling to NF- B: Regulation by Ubiquitination

“…Several polyubiquitination sites have been mapped on NEMO but only Lys-392, the equivalent of human Lys-399, has been tested in a murine knock-in model. These mice were more resistant to lipopolysaccharide (LPS)-induced endotoxic shock but had no defect in T-cell receptor (TCR)-induced proliferation, both of which are mediated by NF-kB signaling (Ni et al 2008). This finding could suggest that ubiquitination of Lys-392 on NEMO is not important for TCR-induced NF-kB activation.…”

NF-κB signaling pathways regulated by CARMA family of scaffold proteins

“…Alternatively, MALT1 may induce NEMO ubiquitination at the Lys399 (Lys392 in mice) residue, because NEMO variant with K399R mutation has been shown to interfere with NF-κB activation [50]. However, mice expressing the NEMO-K392R mutant are not defective in antigen receptorinduced responses [101]. Therefore, it remains to be determined whether MALT1-dependent polyubiquitination of NEMO is functionally important for TCR-induced responses.…”