Cutting edge: K63-linked polyubiquitination of NEMO modulates TLR signaling and inflammation in vivo

Abstract

Transcription factor NF-kappaB controls the expression of multiple genes involved in immunity and inflammation. The initial activation and duration of NF-kappaB signaling is regulated by posttranslational modifications to IkappaB kinase, which earmarks inhibitors of NF-kappaB for degradation. Prior studies suggest that K63-linked ubiquitination of NEMO (NF-kappaB essential modulator), an IkappaB kinase regulatory subunit, is critical for NF-kappaB and MAPK signaling following engagement of Ag receptors. We now demonstrate that NF-kappaB and MAPK pathways are largely unaffected in primary cells from mice harboring a ubiquitination-defective form of NEMO, NEMO-KR. TLR- but not Ag receptor-induced cellular responses are impaired in NEMO-KR mice, which are more resistant to LPS-induced endotoxic shock than wild-type animals. Thus, one function of NEMO ubiquitination is to fine tune innate immune responses under TLR control.

FIGURE 1

Characterization of NEMO-KR mice. A, Structure of the mouse NEMO gene and knock-in alleles. Filled boxes represent NEMO coding exons. The asterisk denotes the Lys→Arg mutation at codon 392. P1 and P2 correspond to probes used for Southern blotting. Restriction enzyme sites: K, KpnI; S, SacI; X, XhoI. Cre-mediated deletion of PGK-Neo from KR-Neo ES cells generated the NEMO-KR allele. B, Southern blot analysis. Probes and restriction fragments are shown in A. The NEMO gene is X-linked and generates only a single band in male ES cells.

FIGURE 2

Impaired humoral immunity in NEMO-KR mice. A, Normal T cell populations in NEMO-KR mice. Splenocytes were stained with Abs for CD4/CD8 and analyzed by FACS. B, T cell proliferation. Purified T cells (10⁶ cells/ml) were cultured with plate-bound anti-CD3 (10 μg/ml), anti-CD3 plus CD28 (10 plus 2 μg/ml, respectively), or not treated (NT). New DNA synthesis was measured by [³H]thymidine uptake. Data in B and C are presented as mean values (±SEM) for three mice (representative of at least two independent experiments). C, B cell proliferation. Purified B cells were cultured with escalating doses of anti-IgM or LPS as indicated (μg/ml) and analyzed as in B. D, Humoral immunity. Mice were immunized with KLH in CFA and sera were collected 2 wk postimmunization. Relative levels of KLH-specific Abs were measure by ELISA. Values represent the mean (±SEM) for four pairs of mice (representative of three independent experiments).

FIGURE 3

Blunted cytokine responses in NEMO-KR cells. A, Cytokine expression. Peritoneal macrophages were cultured with the indicated concentrations of LPS in the presence (IL-12/p40 assays) or absence (IL-6, TNF, and MCP-1 assays) of 30 ng/ml IFN-γ (24 h). Data represent mean cytokine concentrations (±SEM) in culture supernatants from five mice (representative of three independent experiments). B, Peritoneal macrophages were cultured with ligands for TLR2 (heat-killed L. monocytogenes (HKLM); 10⁸ cells/ml), TLR1/2 (tripalmitoylated lipopeptide (Pam); 10 and 100 ng/ml), and TLR 2/6 (N terminus of lipoprotein 44 from M. salivarium (FSL); 10 and 100 ng/ml). After 24 h, IL-6 was measured in culture supernatants as in A. NT, Not treated. C, BMDC were stimulated with LPS (1 ng/ml; 24 h) and cytokines were measured as in A. Data are presented as mean cytokine concentrations from triplicate cultures (±SEM; representative of three independent experiments).

FIGURE 4

NF-κB and MAPK activation in NEMO-KR macrophages. A, Bone marrow macrophages were stimulated with LPS (100 ng/ml for 10 min). Whole cell lysates were boiled in 1% SDS and diluted to 0.1% SDS before NEMO immunoprecipitation (IP). Western blots prepared from NEMO immunocomplexes were probed with anti-Ub Abs (top). A portion of each lysate (5%) was directly blotted (IB) and probed with Abs for NEMO (loading control; middle) or phospho-IκBα (IκBα-P) (activation control; bottom). B and C, Peritoneal macrophages (5 × 10⁶ cells/ml) were stimulated with LPS (100 ng/ml) for the indicated times. Whole cell lysates were analyzed on immunoblots using Abs specific for IκB proteins (B) or phosphorylated (p-) forms of MAPKs (C). Relative levels of β-actin and total Erk provide loading controls.

FIGURE 5

NEMO-KR mice are resistant to LPS-induced endotoxic shock. A, Age-matched WT (n = 6) and NEMO-KR (n = 7) mice were injected with LPS (2 mg per 20 g of body weight). Mortality was assessed daily. Three independent survival studies were performed with similar results (p < 0.05). B, Sera from mice in A were collected at the indicated times. TNF, IL-6, and IFN-γ levels were measured as in Fig. 3 (closed squares, WT; closed triangles, NEMO-KR). Data are presented as mean values (±SEM, representative of two independent experiments).