Genetic variants associated with autoimmunity drive NFκB signaling and responses to inflammatory stimuli

Abstract

The transcription factor nuclear factor κB (NFκB) is a central regulator of inflammation, and genome-wide association studies in subjects with autoimmune disease have identified a number of variants within the NFκB signaling cascade. In addition, causal variant fine-mapping has demonstrated that autoimmune disease susceptibility variants for multiple sclerosis (MS) and ulcerative colitis are strongly enriched within binding sites for NFκB. We report that MS-associated variants proximal to NFκB1 and in an intron of TNFRSF1A (TNFR1) are associated with increased NFκB signaling after tumor necrosis factor-α (TNFα) stimulation. Both variants result in increased degradation of inhibitor of NFκB α (IκBα), a negative regulator of NFκB, and nuclear translocation of p65 NFκB. The variant proximal to NFκB1 controls signaling responses by altering the expression of NFκB itself, with the GG risk genotype expressing 20-fold more p50 NFκB and diminished expression of the negative regulators of the NFκB pathway: TNFα-induced protein 3 (TNFAIP3), B cell leukemia 3 (BCL3), and cellular inhibitor of apoptosis 1 (CIAP1). Finally, naïve CD4 T cells from patients with MS express enhanced activation of p65 NFκB. These results demonstrate that genetic variants associated with risk of developing MS alter NFκB signaling pathways, resulting in enhanced NFκB activation and greater responsiveness to inflammatory stimuli. As such, this suggests that rapid genetic screening for variants associated with NFκB signaling may identify individuals amenable to NFκB or cytokine blockade.

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Figure 1. Naïve CD4 cells from patients with MS exhibit increased phospho-p65 NFκB

Flow cytometry of PBMCs from age-matched healthy control (HC) and relapsing-remitting MS (RRMS) patients stained for CD4, CD45RA, CD45RO, and pS529 p65 NFκB. MFI of p65 results are shown gated on naïve CD4+CD45RA+CD45RO− T-cells. Healthy control n= 34, untreated MS n=11, MS treated n=25. (p-value, unpaired t-test)

Figure 2. MS-associated allelic variants proximal to NFκB1 results in increased TNFα and PMA signaling

A) Association to MS risk in the region surrounding NFκB. Y axis shows the GWAS –log(p value) for the allelic test of association as reported in (12). We have highlighted rs228614 and rs7665090, which are the most associated variants in the region. B) Degradation of IκBα in CD4+CD45RA+CD45RO− T cells after 15 minute TNFα stimulation by flow cytometry in healthy controls. C) phospho-p65 NFκB in CD4+CD45RA+CD45RO− naïve T cells after 15 minute TNFα stimulation by flow cytometry. Results shown normalized to unstimulated T cells. D) rs228614 CD4+CD45RA+CD45RO− T cell degradation of IκBα and phosphorylation of p65 NFκB after 15 minutes of PMA stimulation. E and F) Degradation of IκBα after 15 minutes (E) and phosphorylation of p65 NFκB at 15 and 30 minutes (F), in CD4+CD45RA+CD45RO− T cells after TNFα stimulation by flow cytometry in MS patients. For A–D, rs228614 GG n=4, AG n=18, AA n=6: rs7665090, AA n=8, AG n=49, GG n=23. For E and F, GG n=6, AA n=6.(p value shown for homozygous unpaired t-test)

Figure 3. The rs228614 GG risk genotype results in rapid nuclear localization of p65 NFκB

A) Representative Imagestream data showing nuclear localization of p65 NFκB after 30 minute TNFα stimulation by Amnis Imagestreamx in CD4+ T cells. Green, p65 NFκB. Purple, DAPI. co-localization, overlap of p65 and DAPI by Pearson co-efficient. B) Compilation of rs228614 Nuclear localization of p65 NFκB after TNFα stimulation in CD4+ T cells(GG, n=5, AA n=4). Nuclear localization is shown normalized to unstimulated cells. (p-value shown for unpaired t test).

Figure 4. rs228614 allelic variant results in increased NFκB1 expression and decreased negative regulators of NFκB

A) Representative western blot of p105 and p50 NFκB in total PBMCs and densitometry of total p50 NFκB by western (GG n=7; AA, n=7). B) mRNA expression by Q-PCR of BCL3, TNFAIP3, and CIAP-1 in total PBMCs (GG n=6; AA n=7) by q-PCR. (p-value shown for unpaired t test)

Figure 5. The TNFR1 variant rs1800693 results in increased TNFα responses

A) Association to MS risk in the region surrounding TNFRSF1A. Y axis shows the GWAS –log(p value) for the allelic test of association as reported in (10). We have highlighted rs1800693, the most associated variant in the region. With further replication data in independent samples, rs228614 meets the GWAS significance threshold of p < 5 × 10−8. B) Degradation of IκBα after 30 minutes TNFα stimulation in CD4+CD45RA+CD45RO− T cells. (CC n=14; TT n=20). C) Degradation of IκBα after 30 minutes TNFα stimulation in CD14+ monocytes. (CC n=5; TT n=12). (p-value shown for unpaired t test).

Figure 6. TNFR1 rs1800693 CC risk genotype results in increased NFκB nuclear localization

A) Representative nuclear localization images from TT protective and CC risk genotypes in CD4+ T cells by Amnis Imagstreamx. Green, p65 NFκB. Purple, DAPI. co-localization, overlap of p65 and DAPI by Pearson co-efficient. B) Composite nuclear localization of p65 NFκB after TNFα stimulation in CD4+ T cells (CC n=9; TT n=9). Nuclear localization is shown normalized to unstimulated cells. (p-value shown for unpaired t test).

Figure 7. The TNFR1 variant rs1800693 results in altered intracellular accumulation of TNFR1 and plasma cytokines

A) Confocal microscopy of CD14+ monocytes from the TT (protective) or CC (risk) variant in the TNFR1 region. (blue, DAPI; Green TNFR1). Two of four subjects from each genotype shown. B) Concentration of IL-7, IL-8, GM-CSF, MCP1 and IP10 in plasma samples from healthy control subjects with the CC (risk) or TT (protective) genotypes. (CC n=25; TT n=40). (p value shown for unpaired t test).