A Dynamic Real Time In Vivo and Static Ex Vivo Analysis of Granulomonocytic Cell Migration in the Collagen-Induced Arthritis Model

<div><p>Neutrophilic granulocytes and monocytes (granulomonocytic cells; GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely characterized. Here we have analyzed GMC in the murine collagen-induced arthritis (CIA) model of RA using multi-photon real time in vivo microscopy together with ex vivo analysis of GMC in tissue sections.</p> <p>GMC were abundant as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue. In addition, we observed the frequent formation of cell clusters consisting of both neutrophilic granulocytes and monocytes that actively contributed to the inflammatory process of arthritis. Treatment of animals with a single dose of prednisolone reduced the mean velocity of cell migration and diminished the overall immigration of GMC.</p> <p>In summary, our study shows that the combined application of real time in vivo microscopy together with elaborate static post-mortem analysis of GMC enables the description of dynamic migratory characteristics of GMC together with their precise location in a complex anatomical environment. Moreover, this approach is sensitive enough to detect subtle therapeutic effects within a very short period of time.</p> </div

Kinetics of cell migration.

<p>A) Comparative real time in vivo analysis of cell migration in healthy versus CIA subjects were performed in C57BL/6 lysM-EGFP animals. GMC cells with the expression of EGFP (green) are rarely found in healthy tissue, whereas they are abundant in the synovial tissue of CIA subjects. The magenta signal displays a second harmonic generation (SHG) of collagen structures (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035194#pone.0035194.s002" target="_blank">Movie S1</a>). Scale bars (left corner) represent 100 µm. Representative pictures are shown from movies that were made in three healthy control subjects and three subjects with CIA. B)–D): Quantification of cell velocity (b), displacement over time (c) and the motility index (total displacement/cumulative path length) (d) are shown for GMC cells in healthy subjects, in CIA subjects (CIA) and in CIA subjects treated with prednisolone (CIA+Pred). For (b) and (c), data points represent individual cells (healthy: n = 142; CIA: n = 4055; CIA+Pred: n = 3787) compiled from three independent imaging sessions involving individual joints of three animals. Mean values±s.e.m. are shown. Graph bars show mean±s.e.m. (d). Asterisks (*) indicate significant p values (<0.05). E) Cell immigration describes the number of cells immigrating into the ROI at each time point. Few cells immigrate in healthy subjects. CIA diseased subjects start with high numbers of GMC cells and increasing numbers of cells immigrate into the scanned area. Prednisolone stabilizes cell immigration to comparable numbers of cells immigrating at each time point. Data are mean values from experiment that were performed in three healthy controls, three CIA subjects and three CIA+Prednisolone subjects. Asterisks (*) indicate significant p values (<0.05).</p

Analysis of GMC migration and histomorphological assessment of arthritis in animals with CIA.

<p>A) Paraffin sections were stained with HE and anti-Ly-6B.2 mAb. In contrast to healthy animals GMC cells can be detected in animals with CIA at the earliest time-points (paw swelling day 1) of clinical signs of arthritis and are abundant at later (paw swelling day 10) time-points. Scale bars represent 50 µm. Representative pictures are shown from experiments that were performed in three healthy animals and three animals with CIA. B) Representative samples of paraffin sections from hind paws of three healthy control animals and three animals with CIA stained for HE, TRAP and TB are shown. Arrows point to areas of bone erosions. Scale bars represent 500 µm. C) Histomorphological signs of arthritis were quantified in three CIA subjects (day 11±6 of paw swelling) as compared to three healthy control subjects. Areas of inflammation, bone erosions, osteoclasts and cartilage degradation can be detected in CIA subjects but not in healthy control animals. Graph bars represent mean values ± s.e.m.</p

GMC cluster formation.

<p>A) Real time in vivo cell cluster formation (arrow) during a 2 h time interval. Individual pictures were taken at the indicated time points (Green channel: EGFP⁺ GMC cells, magenta channel: SHG of collagen structures). Scale bars represent 100 µm. Representative pictures are shown from movies that were made in three healthy control subjects and three subjects with CIA. B) Real time in vivo cell cluster formation during a 30 min time interval at higher magnification. Individual pictures of EGFP⁺ cells were taken at the indicated time points. Arrows point at individual cells with high EGFP fluorescence expression intensity. Scale bars represent 15 µm. Representative pictures are shown from movies that were made in three subjects with CIA. Quantification of cell velocity, displacement over time and the motility index (total displacement/cumulative path length) are shown for EGFP^high (defined as 30% of EGFP⁺ cells with highest mean fluorescence intensity of EGFP) and EGFP^low (defined as 30% of EGFP⁺ cells with lowest mean fluorescence intensity of EGFP) cells. Data points represent individual cells (EGFP^high: n = 216; EGFP^low: n = 202) compiled from three independent imaging session from individual joints of three CIA animals. Graph bars show mean±s.e.m. from the same experiments. Asterisks (*) indicate significant p values (p<0.05). Cumulative cell tracks are shown from one representative experiment for EGFP^high, EGFP^low and both (merged) cell populations. Scale bars represent 15 µm.</p

Immigration of GMC over time.

<p>Mean values±s.e.m. were calculated using a linear mixed-effects model including the random factor animal and the interaction between group and time.</p

DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation

<div><p>Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. <i>In vivo</i> this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.</p></div

Loss of ATMIN and NBS1 leads to intestinal inflammation due to infiltration of cytokine-producing T cells.

<p>(A) Histological analysis by H&E staining of large intestine of control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL mice at 12 weeks of age. (B) Histological scores of the large intestine of control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL and 3 individual moribund ATMIN^ΔLNBS1^ΔL mice. (C) Histological analysis by anti-CD3 staining of the large intestine of control and a moribund ATMIN^ΔLNBS1^ΔL mouse. (D) Representative flow cytometry data of CD4 and CD8 expression, as well as (E) TCRβ and TCRγδ expression on isolated IELs from the small intestine of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice, along with the quantification of D-E. N = 5–8 mice per genotype. (F) Representative flow cytometry data of IL17A and IFNγ production by YFP^- and YFP⁺ IELs (gated on the CD4⁺ population) isolated from the small intestine of control and ATMIN^ΔLNBS1^ΔL mice after PMA and ionomycin stimulation. (G) Large intestinal sections from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice were stained for γH2AX and DAPI. Error bars represent SEM (**<i>P</i><0.01, ***<i>P</i><0.001).</p

NBS1 is required for T cell development and TCRα recombination, which is largely unaffected by concomitant loss of ATMIN.

<p>(A) Schematic representation of the strategy to conditionally delete ATMIN and/or NBS1. Exon 4 of ATMIN, exon 6 of NBS1 and a ‘stop’ cassette inserted upstream of the YFP reporter gene (on the Rosa26 locus, denoted as ‘R26’) were flanked by LoxP sites (denoted by arrow heads). Cre recombinase was expressed under the control of the CD2 promotor. (B) Total thymic cellularity and thymic numbers of double-negative (DN; CD4^-CD8^-), double-positive (DP; CD4⁺CD8⁺), CD4 single-positive (SP; CD4⁺CD8^-), CD8 single-positive (SP; CD4^-CD8⁺), TCRβ⁺ (TCRβ⁺ TCRγδ^-) and TCRγδ (TCRβ^- TCRγδ⁺) cells in control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. N = 4–10 mice per genotype. (C) Representative flow cytometry plots for B. (D) Quantitative PCR analysis of eight Vα8-Jα recombination regions in purified DP thymic T cells from control, ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL and ATM^-/- mice. Results are normalized to the control DP thymic T cells. N = 3 mice per genotype. (E) Representative flow cytometry plots of TCRβ⁺HSA^low mature thymocytes isolated from control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. N = 5–7 mice per genotype (F) Quantification of E. (G) Representative flow cytometry data of apoptotic and necrotic (Annexin V⁺ and Annexin V⁺ 7AAD⁺, respectively) DP thymocytes in mice indicated in E. N = 4–7 mice per genotype (H) Quantification of Annexin V⁺ cells within DP, CD4⁺ and CD8⁺ SP thymocytes. Error bars represent SEM (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Loss of ATMIN in combination with NBS1, in T cells, leads to increased mortality due to T cell activation.

<p>(A) Kaplan-Meier survival curve of control, ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL and ATM^-/- mice. Survival was monitored for 32 weeks. (B) Representative images of spleens of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice as well as a moribund ATMIN^ΔLNBS1^ΔL mouse. (C) Histological analysis of the spleen of a control and a moribund ATMIN^ΔLNBS1^ΔL mouse stained for T cells using an anti-CD3 antibody. (D) Representative flow cytometry data of CD4 and CD8 T cells (gated on the TCRβ⁺ population) in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (E) Histological analysis by using an anti-CD3 antibody to visualize T cells in the liver and lung of control and ATMIN^ΔLNBS1^ΔL mice. (F) Representative flow cytometry data of activated CD62L^lowCD44⁺ T cells (gated on the TCRβ⁺ population) in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (G) Quantification of F. N = 3–4 mice per genotype. (H) Flow cytometry data showing the percentage of antigen-experienced CD62L^lowCD4⁺ T cells (gated on the TCRβ⁺ population) in the spleen of control and ATMIN^ΔLNBS1^ΔL mice as well as moribund ATMIN^ΔLNBS1^ΔL mice. (I) Representative flow cytometry data of proliferating (BrdU⁺) T cells (gated on the TCRβ⁺ population) in the spleen of mice indicated in F, measured by <i>in vivo</i> BrdU incorporation over a period of 4 days. (J) Quantification of I. N = 3–4 mice per genotype. Error bars represent SEM (*<i>P</i><0.05, ***<i>P</i><0.001).</p

Loss of ATMIN and NBS1 in T cells leads to the accumulation of DNA damage.

<p>(A) Representative images of FACS sorted (for TCRβ⁺ populations) splenic T cells from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice, analysed using the alkali comet assay. (B) Quantification of A. N = 3 mice per genotype. FACS sorted splenic T cells from NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice were additionally sorted and analysed based on YFP expression. (C) Splenic sections from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice were co-stained for TUNEL and γH2AX. Nuclei were counterstained with DAPI. (D) Western blot analysis of splenic cells from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice for pS15-p53, total p53 and actin. (E) Representative flow cytometry data of CD11b⁺Gr1⁺ neutrophils in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (F) Quantification of E. N = 7–10 mice per genotype. Error bars represent SEM (*<i>P</i><0.05, **<i>P</i><0.01, **** <i>P</i><0.0001).</p