Simple FISH-based evaluation of spermatic nuclear architecture shows an abnormal chromosomal organization in balanced chromosomal rearrangement carriers.

INTRODUCTION: Interphasic DNA has a constant three-dimensional conformation, which is particularly striking for spermatic DNA, with distinct chromosomal territories and a constant chromosomal conformation. We hypothesized that this organization is fragile, and that an excess or a lack of chromosomal segments could hinder the genomic structure as a whole. METHODS: Five human male chromosomal translocation carriers and five controls were included. Spermatic DNA spatial organization was studied, in both balanced and unbalanced spermatozoa, with two-dimensional fluorescent in situ hybridization (FISH) via analysis of chromosomes not implicated in the cases' translocations, compared to that of normal controls. Two parameters were studied: the distance between the two telomeric ends of chromosome 1, and the area of the chromosomal territories of chromosomes 1 and 17. RESULTS: Sperm FISH analysis of rearrangement carriers revealed changes in the nuclear architecture compared to that of controls. Inter-telomeric distance and chromosomal territories areas were both significantly increased. DISCUSSION: We show that an excess or lack of chromosomal segments can hinder the normal spatial nuclear architecture in sperm. These results show that nuclear architecture is a fragile assembly, and that local chromosomal abnormalities may impact the nucleus as a whole. This suggests a potential avenue for selection of spermatozoa prior to in vitro fertilization, not only in rearrangement carriers but also in the infertile population at large. Furthermore, we suggest that 2D-FISH could possibly be a useful tool in assessing spermatic nuclear organization in a way to evaluate male fertility.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

OBJECTIVE: To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). METHODS: The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. RESULTS: Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. CONCLUSION: TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.