We have previously shown that TLR4 triggering promotes the generation of CD23+CD93+ transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23+ transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM+CD23+CD93+ cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM+CD23negCD93+ cells. BAFF-R-Fc–mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-κB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM+CD23+CD93+ cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23+ B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.

The effects of hyperthyroidism on B-cell physiology are still poorly known. In this study, we evaluated the influence of high-circulating levels of 3,5,3 0 -triiodothyronine (T 3 ) on bone marrow, blood, and spleen B-cell subsets, more specifically on B-cell differentiation into plasma cells, in C57BL/6 mice receiving daily injections of T 3 for 14 days. As analyzed by flow cytometry, T 3 -treated mice exhibited increased frequencies of pre-B and immature B-cells and decreased percentages of mature B-cells in the bone marrow, accompanied by an increased frequency of blood B-cells, splenic newly formed B-cells, and total CD19C B-cells. T 3 administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220 C cells correlating with an increased percentage of CD138 C plasma cells was observed in the spleen and bone marrow of T 3 -treated mice. Using enzyme-linked immunospot assay, an increased number of splenic immunoglobulinsecreting B-cells from T 3 -treated mice was detected ex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or together with treatment with T 3 . In conclusion, we provide evidence that high-circulating levels of T 3 stimulate plasmacytogenesis favoring an increase in plasma cells in the bone marrow, a longlived plasma cell survival niche. These findings indicate that a stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.

Ouabain is a steroid described as a compound extracted from plants that is capable of binding to Na+, K+‐ATPase, inhibiting ion transport and triggering cell signaling pathways. Due to its positive ionotropic effect, ouabain was used for more than 200 years for the treatment of cardiac dysfunctions. Numerous antitumor effects of ouabain have been described so far; however, its role on thyroid cancer is still poorly understood. Therefore, the aim of the present work was to evaluate the effect of ouabain on the biology of human papillary thyroid cancer cells. For this, three human thyroid cell lines were used: NTHY‐ori, a non‐tumor lineage, BCPAP and TPC‐1, both derived from papillary carcinomas. Cells were cultured in the presence or absence of ouabain. Subsequently, we evaluated its effects on the viability, cell death, cell cycle, and migratory ability of these cell lines. We also investigated the impact of ouabain in IL‐6/IL‐6R and epithelial to mesenchymal transition markers expression. Our results indicate that ouabain (10−7 M), decreased the number of NTHY‐ori, TPC‐1 and BCPAP viable cells and induced cell cycle arrest after in vitro culture, but did not appear to promote cell death. In TPC‐1 cells ouabain also inhibited cell migration; increased IL‐6/IL‐6R expression and IL‐6 secretion; and diminished vimentin and SNAIL‐1 expression. Collectively, our results indicate that ouabain has an antitumoral role on human papillary thyroid carcinomas in vitro. Even though additional studies are necessary, our work contributes to the discussion of the possibility of new clinical trials of ouabain.