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, 107 (7), 3046-51

A Mutation of Ikbkg Causes Immune Deficiency Without Impairing Degradation of IkappaB Alpha

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A Mutation of Ikbkg Causes Immune Deficiency Without Impairing Degradation of IkappaB Alpha

Owen M Siggs et al. Proc Natl Acad Sci U S A.

Abstract

Null alleles of the gene encoding NEMO (NF-kappaB essential modulator) are lethal in hemizygous mice and men, whereas hypomorphic alleles typically cause a syndrome of immune deficiency and ectodermal dysplasia. Here we describe an allele of Ikbkg in mice that impaired Toll-like receptor signaling, lymph node formation, development of memory and regulatory T cells, and Ig production, but did not cause ectodermal dysplasia. Degradation of IkappaB alpha, which is considered a primary requirement for NEMO-mediated immune signaling, occurred normally in response to Toll-like receptor stimulation, yet ERK phosphorylation and NF-kappaB p65 nuclear translocation were severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond IkappaB alpha degradation, and offers a biochemical explanation for rare immune deficiencies in man.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The panr2 mutation impairs TLR-induced cytokine secretion. Thioglycollate-elicited peritoneal macrophages from WT (n = 12) and panr2 (n = 6) mice were stimulated with TLR ligands, and TNFα, IL-6, IL-12p40, and MCP-1 concentrations were calculated by ELISA. NO was measured by Griess assay, and type I IFN was measured by bioassay. Ligand concentrations were as follows: LPS (800 pg/mL), poly(I:C) (60 μg/mL), Pam3CSK4 (100 ng/mL), R-848 (40 ng/mL), CpG (1 μM), peptidoglycan (PGN, 2 μg/mL), MALP-2 (20 ng/mL). ND, not detected. Bars indicate mean and SE.
Fig. 2.
Fig. 2.
panr2 is a viable allele of Ikbkg. (A) Representative sequence trace of the Ikbkg gene from C57BL/6J and panr2 mice. (B) Genotype ratios of 4-week-old offspring from an Ikbkg+/Y x Ikbkg+/panr2 cross. (C) Expression of NEMO protein in macrophage lysates. (D) Domain structure and location of the panr2 mutation (L153P) in the NEMO protein. H&E staining (E) and mean weight (F) of pooled testes from Ikbkg+/Y and Ikbkgpanr2/Y littermates.
Fig. 3.
Fig. 3.
Impaired development of memory, regulatory and NKT cells. Representative flow cytometry plots and relative frequencies of CD44lo (naive) and CD44hi (memory) T cells in spleen (A), Foxp3+ regulatory T cells in thymus and spleen (B), and CD3ε+NK1.1+ NKT cells in thymus and spleen (C). Absolute numbers are represented in panels DF. Numbers in each flow cytometry plot represents percentages of total lymphocytes, and bars represent mean and SE. (*P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test in DF.)
Fig. 4.
Fig. 4.
Low serum Ig levels and impairment of antibody responses in panr2 mice. (A) Total Ig isotypes in the serum of naive mice as measured by ELISA. Sample concentrations below the detection thresholds for IgG2a and IgA were given values of 12.5 μg/mL and 12.5 ng/mL, respectively. (B) Mice were immunized with a recombinant rSFV-β-gal, and β-gal–specific IgG and IgM at d 14 postimmunization was measured by ELISA.
Fig. 5.
Fig. 5.
The panr2 mutation impairs MAPK and p105 phosphorylation, p65 translocation, and TNF production, but not IκBα degradation. (A) Thioglycollate-elicited macrophages were stimulated with LPS (1 μg/mL) for the indicated times, and cell lysates analyzed by Western blotting. (B) Macrophages were stimulated with LPS, and intracellular (Upper) or surface (Lower) TNF was measured by flow cytometry. (C) Cytoplasmic and nuclear fractions of LPS-stimulated macrophages stimulated with 1 μg/mL LPS were probed with antibodies against NF-κB p65 and β-tubulin.

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