A mutation of Ikbkg causes immune deficiency without impairing degradation of IκBα Owen M. Siggsa, Michael Bergera, Philippe Krebsa, Carrie N. Arnolda, Celine Eidenschenka, Christoph Huberb, Elaine Piriea, Nora G. Smarta, Kevin Khovanantha, Yu Xiaa, Gerald McInerneyc, Gunilla B. Karlsson Hedestamc, David Nemazeeb, and Bruce Beutlera,1 Departments of aGenetics and bImmunology, The Scripps Research Institute, La Jolla, CA 92037; and cDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden Contributed by Bruce Beutler, December 30, 2009 (sent for review December 17, 2009) Null alleles of the gene encoding NEMO (NF-κB essential modulator) Whatever the combination of pathways affected, immune defi- are lethal in hemizygous mice and men, whereas hypomorphic ciency is a unifying consequence of germline NEMO mutations. alleles typically cause a syndrome of immune deficiency and ecto- But of all of the targets of NEMO and the IKK complex, there is no dermal dysplasia. Here we describe an allele of Ikbkg in mice that consensus of which are most critical for immunity. A great deal has impaired Toll-like receptor signaling, lymph node formation, devel- been learned from conditional mutations of IKKα, IKKβ, and opment of memory and regulatory T cells, and Ig production, but did NEMO, but a full understanding of their physiological function κ α not cause ectodermal dysplasia. Degradation of I B , which is con- has been precluded by the embryonic lethality that occurs in their sidered a primary requirement for NEMO-mediated immune signal- absence. The same may be true of the biochemical functions of ing, occurred normally in response to Toll-like receptor stimulation, NEMO, some of which may occur downstream of pathways yet ERK phosphorylation and NF-κB p65 nuclear translocation were arrested in the absence of NEMO (e.g., p65 translocation or severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond TPL2 activation). N N – IκBα degradation, and offers a biochemical explanation for rare Here we describe an -ethyl- -nitrosourea (ENU) induced immune deficiencies in man. mutation of Ikbkg in mice that disrupted TLR signaling and conferred susceptibility to viral and bacterial infection. Hemizygous males were IMMUNOLOGY mutagenesis | N-ethyl-nitrosourea | nuclear factor–κB essential fully viable, yet azoospermic, and most lacked inguinal lymph nodes. modulator | p65 | Toll-like receptor Serum Ig concentrations were reduced, whereas memory, regulatory, and natural killer (NK) T cells were fewer in number. In response to κ α F-κB transcription factors orchestrate numerous immuno- TLR stimulation, I B degradation and phosphorylation of the Nlogical and developmental responses (1). Among the recep- MAPK homologue p38 were intact, yet phosphorylation of ERK and tors that trigger their activity are the Toll-like receptors (TLRs), nuclear translocation of p65 were severely diminished. These data the IL-1 receptor family, B and T cell antigen receptors (BCR/ indicate that broad suppression of NEMO-mediated immune sig- TCR), and TNF receptor family members, including the lym- naling can occur despite IκBα degradation. photoxin β receptor (LTβR), CD40, and ectodysplasin-A receptor (EDAR). NF-κB activity is restrained by members of the IκB Results family, which prevent nuclear translocation of NF-κB subunits. The panr2 Mutation Impairs TLR Signaling and Resistance to Infection. IκB proteins (IκBα,IκBβ, and IκBε) are phosphorylated and Among more than 30,000 third generation (G3) progeny of degraded in response to stimulation, allowing release and nuclear C57BL/6J males mutagenized with ENU, we observed a heritable translocation of NF-κB. IκB phosphorylation is mediated by the phenotype marked by diminished secretion of soluble, bioactive IκB kinase (IKK) complex, which consists of IKK1 (IKKα) and TNF by macrophages stimulated with TLR ligands. This pheno- IKK2 (IKKβ) catalytic subunits, and the regulatory subunit type, designated panr2 (pan-resistance 2), was characterized by an NEMO (IKKγ). impaired response to ligands for TLR3 [poly(I:C)], TLR4 (LPS), IKK activity is also required for phosphorylation of the NF-κB TLR7 (R-848) and TLR9 (CpG oligodeoxynucleotides) and the κ members p105 (NF- B1) and p65 (RelA). Phosphorylated p105 is heterodimers TLR1/2 (Pam3CSK4) and TLR2/6 (MALP-2, pep- polyubiquitinated and degraded in a manner similar to the IκB tidoglycan; Fig. 1). Multiple cytokines were affected, including proteins (2), promoting activation of the TPL2 (tumor progression TNFα, IL-6, IL-12p40, and MCP-1, as well as the inflammatory → locus 2) ERK axis (3, 4). p65 undergoes IKK-dependent mediator NO. Secretion of type 1 IFN in response to TLR3 and fi phosphorylation at serine 536 (5), although the signi cance of this TLR4 stimulation was not affected. panr2 mice were also highly is unclear (1). susceptible to an otherwise sublethal dose of murine cytomega- The range of diseases caused by NEMO mutations highlights lovirus (MCMV; Fig. S1A). Death within 6 d of MCMV infection the physiological importance of NEMO and the IKK complex. was indicative of an innate immune defect, as combined deficiency Null alleles of the X-linked gene encoding NEMO, IKBKG, cause of B and T cells permits survival of a similar infection for more than the inflammatory skin disease incontinentia pigmenti in hetero- 2 weeks (15). Similarly, panr2 mice succumbed to infection with zygous females (6), and are lethal in hemizygous males, as they are Listeria monocytogenes B in mice (7–9). Milder hypomorphic alleles are compatible with within 4 d (Fig. S1 ). viability in males, but cause severe immune deficiency and devel- opmental abnormalities of the teeth, hair, or sweat glands (10). Author contributions: O.M.S. and B.B. designed research; O.M.S., M.B., P.K., C.A., C.E., C. These abnormalities of ectodermal derivatives are thought to H., E.P., K.K., and Y.X. performed research; G.M., G.B.K.H., and D.N. contributed new result from disruption of EDAR signaling, yet there are reports of reagents/analytic tools; O.M.S. and B.B. analyzed data; and O.M.S., N.G.S., and B.B. wrote IKBKG mutations in immune-deficient patients without ecto- the paper. dermal dysplasia (11, 12). Other mutations appear to disrupt The authors declare no conflict of interest. EDAR signaling and CD40-mediated Ig class switching but not 1To whom correspondence should be addressed. E-mail: [email protected]. TLR signaling (13), whereas another mutation disrupts EDAR This article contains supporting information online at www.pnas.org/cgi/content/full/ signaling, but leaves TLR and CD40 signaling largely intact (14). 0915098107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0915098107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 3000 1000 500 wild type panr2 800 400 2000 600 300 (pg/mL) α 400 200 IL-6 (pg/mL) TNF 1000 IL-12p40 (pg/mL) 200 100 ND ND ND ND 0 ND 0 0 4 - 4 N 2 - 4 8 N - 48 -2 :C) K 48 - PS :C) K 4 LPS GN P LPS I S 8 L (I G P-2 CSK CpG P C CpG PG y CS CpG P L 3 R-8 3 R- ALP 3 R-8 A poly(I:C)m MAL poly( M pol m M am a Pa P P 800 100 4000 Fig. 1. The panr2 mutation impairs TLR- 80 induced cytokine secretion. Thioglycollate- 600 3000 elicited peritoneal macrophages from WT (n = nits) /mL) u 12) and panr2 (n =6)micewerestimulated g 60 M) 400 μ 2000 with TLR ligands, and TNFα,IL-6,IL-12p40,and IFN ( IFN -1 (p 1 NO ( 40 MCP-1 concentrations were calculated by e MCP ELISA. NO was measured by Griess assay, and yp 200 t 1000 20 type I IFN was measured by bioassay. Ligand concentrations were as follows: LPS (800 pg/ 0 0 0 mL), poly(I:C) (60 μg/mL), Pam3CSK4 (100 ng/ S ) 4 8 G 2 - S ) 4 G - S 4 G N 2 - μ P C K p - P P K p mL), R-848 (40 ng/mL), CpG (1 M), peptido- L I: P L I:C GN P-2 L I:C) S G P- CS C PGN L CSK Cp P L C C P L 3 R-84 A 3 R-848 A 3 R-848 A μ poly( M poly( M poly( M glycan (PGN,2 g/mL), MALP-2 (20 ng/mL). am P Pam Pam ND, not detected. Bars indicate mean and SE. panr2 Mice Have a Mutation in Ikbkg. Given the broad suppression a T-to-C transition at position 473 of Ikbkg cDNA, in exon 4 of a of NF-κB–dependent cytokine secretion (TNFα, IL-6, IL-12p40, total of 10, leading to a leucine to proline substitution at residue MCP-1), along with normal secretion of an IRF3-dependent 153 (L153P). The affected amino acid lay within the first coiled- cytokine (type 1 IFN), we sequenced the coding exons and splice coil domain of NEMO (Fig. 2D), and did not alter protein junction of five genes known to be required exclusively for TLR- expression or electrophoretic mobility in macrophage lysates (Fig. induced activation of NF-κB(Traf6, Tak1, Ikbkg, Chuk, Ikbkb). Of 2C). Consistent with the chromosomal location of Ikbkg, the panr2 a total of 26,246 target nucleotides, 21,056 (80.2%) were covered phenotype was inherited in an X-linked recessive manner. by high-quality reads in both wild type and mutant templates, and a Unlike Ikbkg knockout mice, panr2 hemizygoteswereviableand single mutation was identified in Ikbkg (Fig. 2A). The mutation was born at Mendelian ratios (Fig. 2B). Conditional deletion of Ikbkg is A C57BL/6J panr2 B male female Ikbkg : +/Y panr2/Y total +/+ +/panr2 total 13 14 27 14 15 29 C Ikbkg+/Y Ikbkg panr2/Y NEMO β-tubulin D IKK binding oligomerization/ubiquitin binding αH1 CC1 αH2 CC2 LZ Pro-rich ZF 63 194 249 278 312 339 389 410 dimerization L153P E Ikbkg+/Y Ikbkgpanr2/Y F 100 Ikbkg+/ Y (n=12) ) Ikbkg panr2/Y (n=8) g 80 m ( t gh i 60 40X we s i t s 40 e Fig.