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. 2016 Oct 18;45(4):903-916.
doi: 10.1016/j.immuni.2016.09.013. Epub 2016 Oct 11.

Deficient Activity of the Nuclease MRE11A Induces T Cell Aging and Promotes Arthritogenic Effector Functions in Patients With Rheumatoid Arthritis

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Deficient Activity of the Nuclease MRE11A Induces T Cell Aging and Promotes Arthritogenic Effector Functions in Patients With Rheumatoid Arthritis

Yinyin Li et al. Immunity. .
Free PMC article

Abstract

Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), T cell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4+ T cells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells. RA T cells had low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage, juxtacentromeric heterochromatin unraveling, and senescence marker upregulation. Inhibition of MRE11A activity in healthy T cells induced the aging phenotype, whereas MRE11A overexpression in RA T cells reversed it. In human-synovium chimeric mice, MRE11Alow T cells were tissue-invasive and pro-arthritogenic, and MRE11A reconstitution mitigated synovitis. Our findings link premature T cell aging and tissue-invasiveness to telomere deprotection and heterochromatin unpacking, identifying MRE11A as a therapeutic target to combat immune aging and suppress dysregulated tissue inflammation.

Conflict of interest statement

The authors declare that no conflict of interest exists.

Figures

Figure 1
Figure 1. Telomeres in RA T cells are shortened and damaged
CD4+CD45RA+ T cells were stimulated for 72 hrs and metaphase nuclei were hybridized with a telomere specific probe (red). DNA is marked with DAPI. (A) Representative microscopy images for one patient with Rheumatoid arthritis (RA) and one healthy control (Con). (B) Fluorescence intensities for 5 patient-control pairs quantified in >30 nuclei for each donor. Fluorescence intensity in arbitrary units (a.u.). Results are mean ± SEM. (C) Distribution of fluorescence intensity (a.u.) strata from 5 RA (red) and 5 control samples (black) quantified in >30 nuclei for each donor. (D) Telomeric ends were analyzed for abnormal structures. Examples of double signal, apposition, fusion, and signal-free ends are shown. (E) Distributions of telomeric phenotypes in 10 patients and 10 controls. 150–200 nuclei were examined in each sample. Percentages of each damage pattern are presented. (F) Naïve and memory CD4+ T cells were separated and placed under proliferative stress by repetitive polyclonal stimulation. Loss of telomeric sequences was measured by PCR and, in parallel, telomeric damage foci were analyzed by dual-color immunostaining with antibodies to the DNA damage protein 53BP1 and the telomeric shelterin TRF2. 53BP1/TRF2 colocalization coefficients and telomeric length shortening in individual samples are correlated. *P < 0.05, two-tailed Student’s t-test. See also Figure S1 and Figure S2.
Figure 2
Figure 2. The aging profile of CD4+CD45RA+ T cells in RA
CD4+CD45RA+ T cells from RA patients and age-matched controls were stimulated for 72 hrs. All data are mean ± SEM. (A, B) CDKN2A, CDKN1A, and TP53 transcript and p16 protein levels were measured in 7 RA-control pairs by RT-PCR and flow cytometry, respectively. (C) Expression of the aging marker CD57 assessed by flow cytometry. (D) SADS foci analyzed by confocal microscopy. Nuclei were hybridized with a sat-II–specific probe (red) and satellite DNA signals were examined in a minimum of 50 nuclei in each sample. Examples of condensed and threadlike distended satellites are shown in inserts. (E) Individual nuclei were scored as SADS-positive, if the pericentromeric or centromeric satellite heterochromatin had lost its tight packaging and was extended longitudinally. Data are from 3 RA-control pairs. (F) Telomeres were uncapped by transfecting healthy CD4+CD45RA+ T cells with TERF2 siRNA oligonucleotides. CDKN2A and CDKN1A transcripts were measured by RT-PCR. Results are from 3 independent experiments. (G) Flow cytometry for p16 expression. Representative histograms from control and TERF2 siRNA transfected cells are shown. Fluorescence Minus One control (FMO) is superimposed as grey area. Mean fluorescence intensities (MFI) of p16 are from 3 independent experiments. (H) CD57 expression was assessed by flow cytometry in 3 independent experiments. Representative data from control and TERF2 siRNA transfected cells are shown.*P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test. See also Figure S3.
Figure 3
Figure 3. MRE11Alow T cells in RA patients
MRE11A protein expression was quantified by intracellular staining of PBMCs in a cohort of RA patients (RA) and age-matched controls (Con) with antibodies against MRE11A and lineage markers (CD4, CD45RA). (A, B) Flow cytometric measurement of MRE11A protein expression in relation to donor age for naïve CD4+CD45RA+ T cells and memory CD4+CD45RO+ T cells. (C) Representative histograms from naïve CD4+CD45RA+ T cells from a healthy individual, an untreated RA patient and a RA patient on therapy. (D) Mean ± SEM of MRE11A MFI are from 5 samples per group. (E, F) Localization of MRE11A to the telomere quantified by dual-color immunostaining with anti-MRE11A (green) and anti-TRF2 (red) in resting naïve CD4+CD45RA+ T cells of 5 RA patients and 5 age-matched controls. DNA is marked with DAPI (blue). (E) Representative image of immunostaining for MRE11A. (F) Staining intensities for total nuclear MRE11A and MRE11A-TRF2 colocalization measured in >50 nuclei from each of 5 different donors. *P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test. See also Figure S4.
Figure 4
Figure 4. Genetic or pharmacologic inhibition of MRE11A induces T cell aging
(A) The impact of MRN insufficiency on telomeric stability was tested by transfecting CD4+CD45RA+ T cells from healthy individuals with control, MRE11A, NBN, or RAD50 siRNA oligonucleotides. 48 hrs later, cells were stained with anti-53BP1 and anti-TRF2. Representative images of 53BP1 (green) and TRF2 (red) after knockdown of individual DNA repair proteins. DNA is marked with DAPI (blue). Merged images show colocalization of 53BP1 and TRF2. (B) Staining intensities (a.u.) for total 53BP1 and 53BP1/TRF2 colocalization were measured in individual nuclei. Mean ± SEM values are indicated. Results are from 5 independent knockdown experiments. (C) T cells were treated with the MRE11A inhibitor Mirin. Damage foci were analyzed by staining for 53BP1 and for 53BP1/TRF2 colocalization. Data are from 5 independent experiments. All data are mean ± SEM. (D, E) CDKN2A and TP53 transcripts were measured by RT-PCR after transfecting CD4+CD45RA+ T cells from healthy individuals with control, MRE11A, NBN, or RAD50 siRNA oligonucleotides for 48 hrs (D) or were treated with the MRE11A inhibitor Mirin (E). (F) Flow cytometry for CD57 expression. Representative data are from control and MRE11A siRNA transfected cells. Percentages of CD57 expressing cells from 3 independent experiments are presented as mean ± SEM. (G) T cells were treated with vehicle or the MRE11A inhibitor Mirin at the indicated doses and analyzed for CD57 expression by flow cytometry. Mean ± SEM from 3 independent experiments. (H, I) Sat-II DNA (red) hybridization in control and Mirin-treated T cells. Distention of satellite DNA in the nuclei of Mirin-treated T cells. Nuclei were scored for SADS as in Figure 2 in >40 nuclei per sample and quantified in 3 control and Mirin-treated samples. *P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test. See also Figure S5.
Figure 5
Figure 5. Overexpression of MRE11A repairs telomeric damage and prevents T cell aging in RA
Naïve CD4+CD45RA+ T cells from RA patients were transfected with control plasmids or a myc-MRE11A construct. After 48 hrs, transfection efficiency was monitored by qPCR (A) and Western blotting (B). The error bars in (A) represent the 95% confidence internal. (C) Representative images of 53BP1 (green) and TRF2 (red) after overexpression of control plasmids or myc-MRE11A. DNA is marked with DAPI (blue). Merged images show colocalization of 53BP1 and TRF2. (D) Staining intensities (a.u.) for 53BP1/TRF2 colocalization were measured in a minimum of 70 individual nuclei from 3 different patients. Mean ± SEM values are indicated. CDKN2A and TP53 transcript levels (E) and p16 protein levels (F) were measured in control (RA+Vec) and myc-MRE11A (RA+MRE11A) transfected cells from 3 different patients. Results are mean ± SEM. (G) Flow cytometry for p16 expression. Representative data are from cells with or without MRE11A overexpression. (H) Percentages of p16 expressing cells analyzed in 4 independent experiments are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test.
Figure 6
Figure 6. The nuclease MRE11A controls pro-arthritogenic effector functions
Synovial inflammation was quantified by gene transcriptional analysis in tissue extracts or by immunohistochemical analysis of tissue sections. (A) TRB transcript levels measured by qPCR to assess T cell accumulation. (B) Representative sections of synovial tissues stained with anti-CD3 antibodies (brown). (C) Percentages of CD3+ T cells in randomly selected fields of synovial tissues sections presented as mean ± SEM. (D) TNFSF11, (E) TNF, IL6, IL1B and (F) TGFB1, IL10 mRNA expression measured by qPCR in tissue extracts. (G) Transcription analysis of the aging markers CDKN2A, CDKN1A, and TP53 by qPCR. (H) p16 (brown) and CD3 (red) were stained by dual-color immunohistochemistry in synovial tissue sections from vehicle and MRE11A inhibitor-treated chimeras. (I) Percentages of p16+CD3+ T cells in randomly selected fields of synovial tissue sections presented as mean ± SEM. (J) T cell migratory capacity was measured in Transwell migration assays in the absence of chemokine gradients. N=6 RA patient-control pairs; n=4 young (<30 yrs) and old (>65 years) healthy individuals; n=4 healthy control T cells with or without Mirin treatment. All data are mean ± SEM.*P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test.
Figure 7
Figure 7. Restoring MRE11A expression in RA T cells prevents pro-arthritogenic effector function
Pairs of NSG mice were engrafted with human synovial tissue and assigned to two treatment arms. CD45ROneg PBMC (A–D) and CD45RAnegPBMC (E–I) were prepared from RA patients and were transfected with either control plasmid or plasmid expressing MRE11A, and adoptively transferred into the chimeric mice. The intensity of synovial inflammation was compared by tissue gene expression analysis applying qPCR. (A, E and G) The density of the synovial T cell infiltrate was captured by TRB and TNFSF11 transcript levels. (B, C and H) Synovial cytokine production capability was assessed through TNF, IL6, IL1B, TGFB1 and IL10 transcript expression. (D, I) The impact of MRE11A overexpression on the tissue presence of aging markers was examined through CDKN2A and TP53 transcript levels. (F) Representative sections of synovial tissues stained with anti-CD3 antibodies (brown). Original magnification, X600 (insets in F). All data are mean ± SEM from at least 6 different synovial grafts.*P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed Student’s t-test. See also Figure S6.

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