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TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

ABSTRACT: In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Common variable immunodeficiency (CVID). The TLR9-mediated survival is enhanced by the vitamin A metabolite retinoic acid (RA). Importantly, neither stimulation of B-cells via TLR9 alone or with RA increases irradiation-induced DNA strand breaks and DNA damage responses such as activation of ATM and DNA-PKcs. We prove that elevated levels of ?H2AX imposed by irradiation of stimulated B-cells is not due to induction of DNA double strand breaks, but merely reflects increased levels of total H2AX upon stimulation. Interestingly however, we unexpectedly find that TLR9 stimulation of B-cells induces low amounts of inactive p53, explained by transcriptional induction of TP53. Taken together, we show that enhanced survival of irradiated B-cells is not accompanied by elevated levels of DNA damage. Our results imply that TLR9-mediated activation of B-cells not only promotes cell survival, but may via p53 provide cells with a barrier against harmful consequences of enhanced activation and proliferation. As CVID-derived B-cells are more radiosensitive and prone to undergo apoptosis than normal B-cells, our data support treatment of CVID patients with CpG-ODN and RA.

SUBMITTER: Holm KL

PROVIDER: S-EPMC5626471 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

Holm Kristine Lillebø KL Syljuåsen Randi Gussgard RG Hasvold Grete G Alsøe Lene L Nilsen Hilde H Ivanauskiene Kristina K Collas Philippe P Shaposhnikov Sergey S Collins Andrew A Indrevær Randi Larsen RL Aukrust Pål P Fevang Børre B Blomhoff Heidi Kiil HK

PloS one 20171003 10

In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Comm ...[more]

PMID: 28973009

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Project description:In common variable immunodeficiency (CVID), heterozygous damaging NFKB1 variants represent the most frequent monogenic cause. NFKB1 encodes the precursor p105, which undergoes proteasomal processing to generate the mature NF-κB transcription factor subunit p50. The majority of NFKB1 sequence changes comprises missense variants of uncertain significance (VUS), each requiring functional evaluation to assess causality, particularly in families with multiple affected members presenting with different phenotypes. In four affected members of a German family, all diagnosed with CVID, we identified a previously uncharacterized heterozygous NFKB1 missense variant (c.1049A>G; p.Tyr350Cys). The clinical phenotypes varied markedly regarding onset, frequency and severity of infections. Consistent immunologic findings were hypogammaglobulinemia with normal specific antibody response to protein- and polysaccharide-based vaccinations, reduced switched memory B cells and decreased lymphocyte proliferation upon stimulation with the B cell mitogen SAC. To assess the pathogenicity of the NFKB1 missense variant, we employed immunophenotyping and functional analyses in a routine in vitro cell culture model. Following site-directed mutagenesis to introduce the variant into overexpression vectors encoding EGFP-fused p105 or p50, we analyzed transiently transfected HEK293T cells by confocal imaging and Western blotting. The cytoplasmic p105-Tyr350Cys precursor gained only weak expression levels indicating accelerated decay. The missense change disabled processing of the precursor to prevent the generation of mutant p50. Unlike the wildtype p50, the overexpressed mutant p50-Tyr350Cys was also not sustainable and showed a conspicuous subnuclear mislocalization with accumulation in dense aggregates instead of a homogenous distribution. Electrophoretic mobility shift assays, fluorescence-based reporter gene analyses and co-transfection experiments however demonstrated, that the DNA-binding activity of p50-Tyr350Cys and the interaction with RelA(p65), IκBα and wildtype p50 were preserved. Mutation carriers had reduced p105 and p50 levels, indicating insufficient protein amounts as the most likely primary defect. In conclusion, the missense variant c.1049A>G caused a detrimental defect, preventing the persistent expression of both, the p105-Tyr350Cys precursor and the mature p50-Tyr350Cys. The variable clinical phenotypes among affected family members sharing an identical pathogenic NFKB1 variant support a disease mechanism provoked by a p105/p50 (haplo)insufficient condition.

Dataset Information

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

Publications

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

Similar Datasets

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