Research ArticleMultiple Sclerosis

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

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Science Translational Medicine  10 Jun 2015:
Vol. 7, Issue 291, pp. 291ra93
DOI: 10.1126/scitranslmed.aaa9223
  • Fig. 1. Naïve CD4 cells from patients with MS exhibit increased phospho-p65 NFκB.

    Flow cytometry of PBMCs from age-matched healthy control (HC) and RRMS patients stained for CD4, CD45RA, CD45RO, and pS529 p65 NFκB. Mean fluorescence intensity (MFI) results of p65 are shown gated on naïve CD4+CD45RA+CD45RO T cells. Healthy control, n = 34; untreated MS, n = 11; treated MS, n = 25. P value shown for unpaired t test. n.s., not significant.

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

    (A) Association with 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. cM, centimorgan. (B) Degradation of IκBα in CD4+CD45RA+CD45RO T cells after 15 min of TNFα stimulation by flow cytometry in healthy controls. (C) Phospho-p65 NFκB in CD4+CD45RA+CD45RO naïve T cells after 15 min of 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 min of PMA stimulation. (E and F) Degradation of IκBα after 15 min (E) and phosphorylation of p65 NFκB at 15 and 30 min (F) in CD4+CD45RA+CD45RO T cells after TNFα stimulation by flow cytometry in MS patients. For (A) to (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.

  • Fig. 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 min of TNFα stimulation by Amnis Imagestreamx in CD4+ T cells. Green, p65 NFκB; purple, DAPI; colocalization, overlap of p65 and DAPI by Pearson coefficient. (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.

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

    (A) Representative Western blot of p105 and p50 NFκB in total PBMCs and densitometry of total p50 NFκB by Western blot (GG, n = 7; AA, n = 7). (B) mRNA expression by quantitative polymerase chain reaction (qPCR) of BCL3, TNFAIP3, and CIAP1 in total PBMCs (GG, n = 6; AA, n = 7). P value shown for unpaired t test.

  • Fig. 5. The TNFR1 variant rs1800693 results in increased TNFα responses.

    (A) Association with 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 min of TNFα stimulation in CD4+CD45RA+CD45RO T cells (CC, n = 14; TT, n = 20). (C) Degradation of IκBα after 30 min of TNFα stimulation in CD14+ monocytes (CC, n = 5; TT, n = 12). P value shown for unpaired t test.

  • Fig. 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 Imagestreamx. Green, p65 NFκB; purple, DAPI; colocalization, overlap of p65 and DAPI by Pearson coefficient. (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.

  • Fig. 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 are shown. (B) Concentrations 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.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/291/291ra93/DC1

    Fig. S1. Confirmation of increased pNFκB in MS patients.

    Fig. S2. Gating strategy and representative histogram for rs228614 GG (risk) and AA (protective) variants.

    Fig. S3. rs228614 and rs7665090 proximal to NFκB1 result in increased IκBα degradation after TNFα treatment.

    Fig. S4. Constitutive total IκBα and phospho-p65 NFκB are similar in rs228614 risk and protective variants.

    Fig. S5. Full uncut gel for Fig. 4.

    Fig. S6. TNFα and PMA responses are stable across multiple draws.

    Fig. S7. Responses to TNFα do not change with age, gender, or ethnicity.

    Fig. S8. TNFα signals through TNFR1 on naïve CD4 cells.

    Fig. S9. The rs1800693 CC variant does not change the surface expression of TNFR1.

    Fig. S10. Representative single-stained control.

    Table S1. Demographics of healthy subjects and MS samples for Fig. 1 and fig. S1.

    Table S2. Demographics of rs228614 results from Fig. 2.

    Table S3. Demographics of rs7665090 from Fig. 2.

    Table S4. Demographics of rs1800693 from Fig. 5.

    Table S5. Luminex data from rs1800693 subjects.

    Table S6. Raw data.

  • Supplementary Material for:

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

    William J. Housley, Salvador D. Fernandez, Kenneth Vera, Sasidhar R. Murikinati, Jaime Grutzendler, Nicole Cuerdon, Laura Glick, Phillip L. De Jager, Mitja Mitrovic, Chris Cotsapas, David A. Hafler*

    *Corresponding author. E-mail: david.hafler{at}yale.edu

    Published 10 June 2015, Sci. Transl. Med. 7, 291ra93 (2015)
    DOI: 10.1126/scitranslmed.aaa9223

    This PDF file includes:

    • Fig. S1. Confirmation of increased pNFκB in MS patients.
    • Fig. S2. Gating strategy and representative histogram for rs228614 GG (risk) and AA (protective) variants.
    • Fig. S3. rs228614 and rs7665090 proximal to NFκB1 result in increased IκBα degradation after TNFα treatment.
    • Fig. S4. Constitutive total IκBα and phospho-p65 NFκB are similar in rs228614 risk and protective variants.
    • Fig. S5. Full uncut gel for Fig. 4.
    • Fig. S6. TNFa and PMA responses are stable across multiple draws.
    • Fig. S7. Responses to TNFa do not change with age, gender, or ethnicity.
    • Fig. S8. TNFa signals through TNFR1 on naïve CD4 cells.
    • Fig. S9. The rs1800693 CC variant does not change the surface expression of TNFR1.
    • Fig. S10. Representative single-stained control.
    • Table S1. Demographics of healthy subjects and MS samples for Fig. 1 and fig. S1.
    • Table S2. Demographics of rs228614 results from Fig. 2.
    • Table S3. Demographics of rs7665090 from Fig. 2.
    • Table S4. Demographics of rs1800693 from Fig. 5.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Luminex data from rs1800693 subjects.
    • Table S6 (Microsoft Excel format). Raw data.

    [Download Table S5 and S6]

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