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Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα.

Mooster JL, Le Bras S, Massaad MJ, Jabara H, Yoon J, Galand C, Heesters BA, Burton OT, Mattoo H, Manis J, Geha RS - J. Exp. Med. (2015)

Bottom Line: Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID.Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling.IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs.

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Affiliation: Division of Allergy and Immunology and Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Division of Transfusion Medicine, and Department of Pathology, Harvard Medical School, Boston, MA 02115.

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IκBα mutant mice have ED, impaired IκBα processing, and deficient TLR response. (A) IκBα mutant mouse and WT littermate photographed at 3 wk of age. Data are representative of >20 mice per group. (B and C) Growth (B) and Kaplan-Meier survival (C) curves of IκBα mutant mice and WT littermates weighed every 3–4 d and observed daily. Data were derived from 34 mutant mice and 19 WT littermates weighed. (D–F) Photographs of mandibular bones (D) and fur (E) and H&E staining of footpad sections (F) in 6 wk-old IκBα mutant mouse and WT littermate. Red arrows in D show the missing third molars in the mutant mice. Data are representative of four or more mice per group in three independent experiments. Bar, 100 µm. (G) Immunoblot of fibroblast lysates using antibodies to phospho-IκBα or IκBα. Actin was used as a loading control. Pooled results of IκBα phosphorylation at 5 min using three mice per group in three independent experiments. (H) TNF as measured by ELISA in supernatants of BMDCs stimulated with the TLR ligands PamCsk4 (TLR1/2), LPS (TLR4), loxoribine (TLR7), and CpG oligonucleotide (TLR9). Data are representative of four mice per group in two independent experiments for PamCsk4 and LPS and four mice per group in three independent experiments for loxoribine and CpG. (I and J) qPCR analysis of Vcam1 and Icam1 mRNA (I) and flow cytometric analysis of VCAM1 surface expression (J) after TNF stimulation in MEFs from IκBα mutant mice and WT controls. Results in I are expressed relative to unstimulated WT MEFs. Data are representative of three mice per group in three independent experiments. Circles and columns represent means, and bars represent SD in B, C, and G–I. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig1: IκBα mutant mice have ED, impaired IκBα processing, and deficient TLR response. (A) IκBα mutant mouse and WT littermate photographed at 3 wk of age. Data are representative of >20 mice per group. (B and C) Growth (B) and Kaplan-Meier survival (C) curves of IκBα mutant mice and WT littermates weighed every 3–4 d and observed daily. Data were derived from 34 mutant mice and 19 WT littermates weighed. (D–F) Photographs of mandibular bones (D) and fur (E) and H&E staining of footpad sections (F) in 6 wk-old IκBα mutant mouse and WT littermate. Red arrows in D show the missing third molars in the mutant mice. Data are representative of four or more mice per group in three independent experiments. Bar, 100 µm. (G) Immunoblot of fibroblast lysates using antibodies to phospho-IκBα or IκBα. Actin was used as a loading control. Pooled results of IκBα phosphorylation at 5 min using three mice per group in three independent experiments. (H) TNF as measured by ELISA in supernatants of BMDCs stimulated with the TLR ligands PamCsk4 (TLR1/2), LPS (TLR4), loxoribine (TLR7), and CpG oligonucleotide (TLR9). Data are representative of four mice per group in two independent experiments for PamCsk4 and LPS and four mice per group in three independent experiments for loxoribine and CpG. (I and J) qPCR analysis of Vcam1 and Icam1 mRNA (I) and flow cytometric analysis of VCAM1 surface expression (J) after TNF stimulation in MEFs from IκBα mutant mice and WT controls. Results in I are expressed relative to unstimulated WT MEFs. Data are representative of three mice per group in three independent experiments. Circles and columns represent means, and bars represent SD in B, C, and G–I. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: The strategy for the generation and identification of the heterozygous IκBα S32I mutant (IκBα mutant) mice is shown in Fig. S1. IκBα mutant mice were born at the normal Mendelian ratio but were significantly smaller in size and weight than their WT littermates (Fig. 1, A and B) and had a 50% survival rate at 8 wk compared with 100% for WT littermates (Fig. 1 C). IκBα mutant mice are missing their third molars, lack guard hairs, and have hypoplastic eccrine glands (Fig. 1, D–F), a phenotype observed in mice with disruption of the Eda gene, mutated in patients with X-linked anhidrotic ED (Srivastava et al., 2001).


Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα.

Mooster JL, Le Bras S, Massaad MJ, Jabara H, Yoon J, Galand C, Heesters BA, Burton OT, Mattoo H, Manis J, Geha RS - J. Exp. Med. (2015)

IκBα mutant mice have ED, impaired IκBα processing, and deficient TLR response. (A) IκBα mutant mouse and WT littermate photographed at 3 wk of age. Data are representative of >20 mice per group. (B and C) Growth (B) and Kaplan-Meier survival (C) curves of IκBα mutant mice and WT littermates weighed every 3–4 d and observed daily. Data were derived from 34 mutant mice and 19 WT littermates weighed. (D–F) Photographs of mandibular bones (D) and fur (E) and H&E staining of footpad sections (F) in 6 wk-old IκBα mutant mouse and WT littermate. Red arrows in D show the missing third molars in the mutant mice. Data are representative of four or more mice per group in three independent experiments. Bar, 100 µm. (G) Immunoblot of fibroblast lysates using antibodies to phospho-IκBα or IκBα. Actin was used as a loading control. Pooled results of IκBα phosphorylation at 5 min using three mice per group in three independent experiments. (H) TNF as measured by ELISA in supernatants of BMDCs stimulated with the TLR ligands PamCsk4 (TLR1/2), LPS (TLR4), loxoribine (TLR7), and CpG oligonucleotide (TLR9). Data are representative of four mice per group in two independent experiments for PamCsk4 and LPS and four mice per group in three independent experiments for loxoribine and CpG. (I and J) qPCR analysis of Vcam1 and Icam1 mRNA (I) and flow cytometric analysis of VCAM1 surface expression (J) after TNF stimulation in MEFs from IκBα mutant mice and WT controls. Results in I are expressed relative to unstimulated WT MEFs. Data are representative of three mice per group in three independent experiments. Circles and columns represent means, and bars represent SD in B, C, and G–I. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig1: IκBα mutant mice have ED, impaired IκBα processing, and deficient TLR response. (A) IκBα mutant mouse and WT littermate photographed at 3 wk of age. Data are representative of >20 mice per group. (B and C) Growth (B) and Kaplan-Meier survival (C) curves of IκBα mutant mice and WT littermates weighed every 3–4 d and observed daily. Data were derived from 34 mutant mice and 19 WT littermates weighed. (D–F) Photographs of mandibular bones (D) and fur (E) and H&E staining of footpad sections (F) in 6 wk-old IκBα mutant mouse and WT littermate. Red arrows in D show the missing third molars in the mutant mice. Data are representative of four or more mice per group in three independent experiments. Bar, 100 µm. (G) Immunoblot of fibroblast lysates using antibodies to phospho-IκBα or IκBα. Actin was used as a loading control. Pooled results of IκBα phosphorylation at 5 min using three mice per group in three independent experiments. (H) TNF as measured by ELISA in supernatants of BMDCs stimulated with the TLR ligands PamCsk4 (TLR1/2), LPS (TLR4), loxoribine (TLR7), and CpG oligonucleotide (TLR9). Data are representative of four mice per group in two independent experiments for PamCsk4 and LPS and four mice per group in three independent experiments for loxoribine and CpG. (I and J) qPCR analysis of Vcam1 and Icam1 mRNA (I) and flow cytometric analysis of VCAM1 surface expression (J) after TNF stimulation in MEFs from IκBα mutant mice and WT controls. Results in I are expressed relative to unstimulated WT MEFs. Data are representative of three mice per group in three independent experiments. Circles and columns represent means, and bars represent SD in B, C, and G–I. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: The strategy for the generation and identification of the heterozygous IκBα S32I mutant (IκBα mutant) mice is shown in Fig. S1. IκBα mutant mice were born at the normal Mendelian ratio but were significantly smaller in size and weight than their WT littermates (Fig. 1, A and B) and had a 50% survival rate at 8 wk compared with 100% for WT littermates (Fig. 1 C). IκBα mutant mice are missing their third molars, lack guard hairs, and have hypoplastic eccrine glands (Fig. 1, D–F), a phenotype observed in mice with disruption of the Eda gene, mutated in patients with X-linked anhidrotic ED (Srivastava et al., 2001).

Bottom Line: Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID.Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling.IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Allergy and Immunology and Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Division of Transfusion Medicine, and Department of Pathology, Harvard Medical School, Boston, MA 02115.

Show MeSH
Related in: MedlinePlus