Research ArticleAutoimmunity

Impaired ATM activation in B cells is associated with bone resorption in rheumatoid arthritis

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Science Translational Medicine  20 Nov 2019:
Vol. 11, Issue 519, eaaw4626
DOI: 10.1126/scitranslmed.aaw4626
  • Fig. 1 Relative ATM insufficiency identifies a subgroup of patients with RA.

    (A) Two groups of patients with RA: group I and group II identified on the basis of differential expression of ATM and ATM-related genes amplified from 1 × 105 to 3 × 105 bulk-sorted CD19+CD21+CD27 naïve B cells. Gene array of genes in the ATM-regulated response to DNA DSB with at least twofold difference in expression between group I and group II patients. Lower expression (green); higher expression (orange). IDs of patients with RA are indicated. (B) Overlap between genes differentially regulated in group I versus group II patients with RA. Curated gene sets (blue dots) from the Molecular Signatures Database whose transcripts correlate significantly with ATM expression by Pearson correlation coefficient (ATM PCC), BRCA1 expression (BRCA1 PCC), genes induced or repressed by the DNA DSB–inducing agent doxorubicin (DOX up, DOX dn), genes induced in response to UV DNA damage (UV diff and UV late), and genes induced by overexpression of p53 (P53 up). Gray dots represent other gene sets with significant overlaps that do not relate to the ATM cascade. (C) Pathway analysis of genes that are differentially regulated in B cells from group I relative to group II patients with RA. Senescence pathway (SEN), cell stress pathway (STR), cell cycle checkpoint pathway (CCC), death receptors and ligands pathway (DRL), and DNA damage sensing and response pathway (DDR).

  • Fig. 2 Skewed Igκ repertoire in patients with RA who have decreased ATM function.

    (A) Summary of Jκ1 gene usage frequency (HD, open diamond; combined RA, black diamonds, same subjects as shown in blue and green; group I RA, blue diamonds; group II RA, green diamonds; and patients with AT, blue squares). (B) Upstream Vκ gene usage frequency was plotted against downstream Jκ3-4-5 gene usage in transitional B cells from 25 HD controls, 6 patients with AT, and 43 patients with RA. (C) Flow cytometry performed on CD20+ B cells from a representative HD control, group I patient with RA (RA-R01), group II patient with RA (RA-R04), and a patient with AT (AT-Y01) for analysis of activated pATM and presence of DNA DSB (induction of γH2AX) with (gray histograms) or without (open histograms) x-ray irradiation. (D) Summary of pATM fold changes in B cells from HD (open diamonds), patients with RA (combined RA, black diamonds; group I, blue diamonds; group II, green diamonds), and patients with AT (blue squares). Each diamond/square represents an individual or patient, and bars represent mean value. Mann-Whitney U testing for difference between groups, and P values are indicated when significant.

  • Fig. 3 Increased frequencies of circulating atypical CD21−/lo B cells correlate with high prevalence of joint erosion in group I patients with RA.

    (A) Representative flow cytometry histograms on gated CD19+CD10CD27 B cells from peripheral blood of an HD, group I and group II patients with RA, and a patient with AT for evaluating CD21−/lo B cell frequency. (B) Summary of frequencies of circulating CD21−/lo B cells among CD19+CD10CD27 B cells in HD (open diamonds), group I (blue diamonds), group II (green diamonds), and AT (light blue squares). Each symbol represents an individual. Mann-Whitney U testing for difference between groups, and P values are indicated when significant. (C) Relative fold change in gene expression for chemokine and cytokine receptors in CD19+CD10CD27CD21−/lo relative to conventional CD19+CD10CD27CD21+ naïve B cells from group I patients with RA. (D) Prevalence of erosive joint disease among group I and group II patients. Erosive disease was assessed by the patient’s rheumatologist and indicated present or absent. (E) Frequency of patients without joint erosions in group I versus group II. Prevalence of erosive joint disease compared with χ2 testing for difference in proportions between groups. *Significance with P < 0.05.

  • Fig. 4 ATM inhibition induces a pro-osteoclastogenic phenotype in human B cells.

    Cytokine gene transcripts assessed by qPCR (A) and secreted protein assessed by enzyme-linked immunosorbent assay (ELISA) (B) in CD20+ B cells from HD cultured in the presence or absence of 10 μM KU55933 (ATMi). Dashed line represents unstimulated baseline. *Significant fold induction over unstimulated baseline at P < 0.05 with two-tailed t testing. (C) Effect of ATM inhibition on the ability of CD40L and αIgM stimulation to induce RANKL and OPG protein expression. Comparison by two-tailed t testing relative to culture with CD40L and αIgM alone (dashed line), * and ‡ represent significant difference relative to cultures with CD40L and αIgM alone. Error bars, SEM for all graphs. (D) RANKL:OPG ratio in different culture conditions as noted. Bars represent mean ratios of the aggregated expression from five individual HDs. Comparisons between conditions tested by analysis of variance (ANOVA) with multiple comparisons, and P values are indicated when significant.

  • Fig. 5 In vivo ATM inhibition recapitulates skewed Igκ repertoire.

    (A) Representative flow cytometry histograms for γH2AX induction on the top panels for human transitional B cells from a control (top left) and ATMi-injected NSG humanized mice (top right). Representative flow cytometry histograms for activated pATM on the bottom panels. (B) Summary of pATM fold changes. Each diamond represents an NSG humanized mouse (open, control; filled, injected with ATMi), and bars represent mean value. Mann-Whitney U testing for differences between groups. (C) Summary of upstream Vκ gene segment usage in human transitional B cells isolated from NSG humanized mice; bars represent mean value. Mann-Whitney U testing for differences between groups. (D) Representative graphs of individual Jκ gene segment usage proportions in human transitional B cells from control and ATMi-injected NSG humanized mice. (E) Upstream Vκ gene usage frequency was plotted against that of downstream Jκ3-4-5 gene usage in human transitional B cells from NSG humanized mice with or without ATMi injections. Data were obtained by analyzing human B cells isolated from five controls and five NSG humanized mice injected with ATMi. Each diamond represents a mouse (open, control; filled, injected with ATMi). Human HSCs were obtained from five different human fetal donors and transplanted into five different sibling pairs of mice. Each murine sibling pair was born at the same time from the same mother and cohoused for life. Data represent experiments on five separate murine sibling pairs from five separate litters from five separate murine mothers. P values are indicated when significant.

  • Fig. 6 ATM inhibition does not impair secondary recombination.

    (A) Representative flow cytometry plots of κ:λ staining on peripheral transitional B cells from control NSG and ATMi injected (NSG + ATMi injection) humanized mice. Summary of κ:λ ratio compared between NSG control and NSG + ATMi injection. (B) κDEs and KRECs PCRs in Igλ+ transitional human B cells from NSG humanized mice without (control) or with ATMi injection. Cycle threshold (CT) value to reach threshold is depicted. (C) Representative flow cytometry plots of CD69 and CD10 in bone marrow B cells from control NSG and NSG + ATMi injection humanized mice. Bar graph depicts fold change in frequency of CD10+CD69+ B cells. (D) Comparison of average mean fluorescence intensity (MFI) of CXCR4 in CD69+ pre–B cells and immature B cells in NSG control and NSG mice injected with ATMi. (E) Representative flow cytometry plots of annexin V versus 7AAD staining in CD10+CD34IgM+ B cells. Bar graph represents average fold change in frequency of annexin V+ among CD10+CD34IgM+ B cells. (F) CD19+ B cells in the bone marrow of ATMi-treated (NSG + ATMi) compared with matched control NSG humanized mice. All experiments represent n = 4 or 5 mice per group. Error bars, SEM. Paired two-tailed t testing, and P values are indicated when significant.

  • Fig. 7 Decreased bone density after ATM inhibition in humanized mice.

    Average bone tissue density in (A) cortex and (B) trabeculae. (C) Average cortical thickness and (D) ratios of cortical bone volume to tissue volume (BV/TV) obtained by μCT performed on tibiae from nonhumanized control and ATMi-injected NSG mice (open bars) and control and ATMi-injected NSG humanized mice (filled bars). Comparisons were made via paired t testing with error bars indicating SEM, and P values are indicated when significant.

  • Fig. 8 Increased B cell RANKL relative to OPG in humanized mice with decreased bone density.

    (A) Representative flow cytometry plot showing CD40 expression in human CD19+CD10+CD34 bone marrow B cells developing in control and ATMi-injected NSG humanized mice (left panel). Right panel summarizes fold changes in % CD40+ cells from control (open bar) and ATMi-injected (filled bar) mice. (B) Representative flow cytometry plot showing RANKL expression on gated bone marrow CD19+CD10+CD34IgM+ B cells in control and ATMi-injected NSG humanized mice (left panel). Right panel shows RANKL MFI in RANKL+ B cells from control (open bar) and ATMi-injected (filled bar) NSG humanized mice. OPG (C) and RANKL (D) production measured by ELISA from in vitro cultures of human bone marrow B cells isolated from control and ATMi-injected NSG humanized mice and stimulated with CD40L and anti-IgM for 48 hours. (E) Comparison of fold change in RANKL and OPG in bone marrow B cells from ATMi-injected relative to control NSG humanized mice. *Significant fold change (P < 0.05). Comparisons were made via paired t testing with error bars indicating SEM, and P values are indicated when significant.

  • Table 1 Characteristics of group I and group II patients with RA.

    Mann-Whitney U testing for difference between groups except prior DMARD use, which was compared by χ2 for equality in proportions. Significance at P < 0.05.

    Group I
    (n = 18)
    Group II
    (n = 25)
    P value
    Age (years ± SD)55 ± 14.750 ± 11.70.55
    Disease
    duration
    (years ± SD)
    3 ± 7.22 ± 6.90.45
    Prior DMARD
    use*
    (number of
    patients)
    990.36
    RF titer ± SD387 ± 397.4268 ± 307.80.35
    Anti-CCP
    antibody
    titer ± SD
    142 ± 86.5178 ± 88.10.36

    *Includes conventional oral DMARDs and biologics/small-molecule inhibitors with the exception of the anti-CD20+ B cell–depleting biologic rituximab.

    Supplementary Materials

    • stm.sciencemag.org/cgi/content/full/11/519/eaaw4626/DC1

      Fig. S1. Skewed Igκ repertoire in patients with RA and decreased ATM function.

      Fig. S2. Group I and group II patients with RA display similar biometric characteristics.

      Fig. S3. Decreased pATM induction in T cells from patients with RA.

      Fig. S4. Decreased pATM induction in non–T and non–B cells from patients with RA.

      Fig. S5. In vitro inhibition of ATM activation in human B cells.

      Fig. S6. Reconstitution of human lymphocytes in NSG humanized mice.

      Fig. S7. B cell precursors are the major human CD45+ cell population in the bone marrow of NSG humanized mice.

      Fig. S8. Trabecular thickness and ratios of bone volume to tissue volume in NSG humanized mice injected or not with ATMi.

      Table S1. Igκ repertoire of antibodies expressed by new emigrant/transitional B cells from patients with RA*, patients with AT, and HDs*.

      Table S2. Additional characteristics of group I and group II patients with RA.

      Table S3. Igκ repertoire of antibodies expressed by new emigrant/transitional B cells from NSG humanized mice.

      Data file S1. Primary data.

    • The PDF file includes:

      • Fig. S1. Skewed Igκ repertoire in patients with RA and decreased ATM function.
      • Fig. S2. Group I and group II patients with RA display similar biometric characteristics.
      • Fig. S3. Decreased pATM induction in T cells from patients with RA.
      • Fig. S4. Decreased pATM induction in non–T and non–B cells from patients with RA.
      • Fig. S5. In vitro inhibition of ATM activation in human B cells.
      • Fig. S6. Reconstitution of human lymphocytes in NSG humanized mice.
      • Fig. S7. B cell precursors are the major human CD45+ cell population in the bone marrow of NSG humanized mice.
      • Fig. S8. Trabecular thickness and ratios of bone volume to tissue volume in NSG humanized mice injected or not with ATMi.
      • Table S1. Igκ repertoire of antibodies expressed by new emigrant/transitional B cells from patients with RA*, patients with AT, and HDs*.
      • Table S2. Additional characteristics of group I and group II patients with RA.
      • Table S3. Igκ repertoire of antibodies expressed by new emigrant/transitional B cells from NSG humanized mice.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

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