Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Lithium impacts the function of hematopoietic stem cells via disturbing the endoplasmic reticulum stress and Hsp90 signaling.

Lithium (Li) has been widely used in clinical therapy and new Li-ion battery industry. To date, the impact of Li on the development of immune cells is largely unknown. The aim of this study was to investigate the impact of Li on hematopoiesis. C57BL/6 (B6) mice were treated with 50 ppm LiCl, 200 ppm LiCl, or the control via drinking water for 3 months, and thereafter the hematopoiesis was evaluated. Treatment with Li increased the number of mature lymphoid cells while suppressing the number of mature myeloid cells in mice. In addition, a direct action of Li on hematopoietic stem cells (HSC) suppressed endoplasmic reticulum (ER) stress to reduce the proliferation of HSC in the bone marrow (BM), thus leading to fewer HSC in mice. On the other hand, the suppression of ER stress by Li exposure increased the expression of Hsp90, which promoted the potential of lymphopoiesis but did not impact that for myelopoiesis in HSC in the BM of mice. Moreover, in vitro treatment with Li also likely disturbed the ER stress-Hsp90 signaling, suppressed the proliferation, and increased the potential for lymphopoiesis in human HSC. Our study reveals a previously unrecognized toxicity of Li on HSC and may advance our understanding for the immunotoxicology of Li.

Lead exposure suppresses the Wnt3a/ß-catenin signaling to increase the quiescence of hematopoietic stem cells via reducing the expression of CD70 on bone marrow-resident macrophages.

Lead (Pb) is a heavy metal highly toxic to human health in the environment. The aim of this study was to investigate the mechanism of Pb impact on the quiescence of hematopoietic stem cells (HSC). WT C57BL/6 (B6) mice treated with 1250 ppm Pb via drinking water for 8 weeks had increased the quiescence of HSC in the bone marrow (BM), which was caused by the suppressed activation of the Wnt3a/ß-catenin signaling. Mechanically, a synergistic action of Pb and IFNγ on BM-resident macrophages (BM-Mφ) reduced their surface expression of CD70, which thereby dampened the Wnt3a/ß-catenin signaling to suppress the proliferation of HSC in mice. In addition, a joint action of Pb and IFNγ also suppressed the expression of CD70 on human Mφ to impair the Wnt3a/ß-catenin signaling and reduce the proliferation of human HSC purified from umbilical cord blood of healthy donors. Moreover, correlation analyses showed that the blood Pb concentration was or tended to be positively associated with the quiescence of HSC, and was or tended to be negatively associated with the activation of the Wnt3a/ß-catenin signaling in HSC in human subjects occupationally exposed to Pb. Collectively, these data indicate that an occupationally relevant level of Pb exposure suppresses the Wnt3a/ß-catenin signaling to increase the quiescence of HSC via reducing the expression of CD70 on BM-Mφ in both mice and humans.

Cadmium impairs the development of natural killer cells and bidirectionally modifies their capacity for cytotoxicity.

Cadmium (Cd) is a highly toxic heavy metal in the environment. The aim of this study was to investigate the impact of Cd on natural killer (NK) cells. C57BL/6 mice were treated with 10 ppm Cd via drinking water for 3 months, and the development of NK cells in the bone marrow (BM) and the cytotoxicity of mature NK (mNK) cells in the peripheral immune organs were evaluated thereafter; the impact of Cd on the cytotoxicity of mNK cells from human peripheral blood mononuclear cells (PBMC) was also investigated. Whereas Cd treatment impaired the differentiation of NK progenitors in the BM, Cd treatment activated the JAK3/STAT5 signaling to drive the proliferation of mNK cells and thereby lead to a compensation increase of mNK cells in the peripheral immune organs of mice. Additionally, Cd treatment bidirectionally regulated the cytotoxicity of mouse mNK cells to differential tumor cells, dependent on the levels of Fas expression in the tumor cells; mechanically, Cd treatment activated the JAK3/STAT5 signaling to promote the expression of FasL in mNK cells to increase their cytotoxicity, while Cd treatment reduced the expression of granzyme B in mNK cells to impair their cytotoxicity in the peripheral immune organs of mice. Likewise, in vitro assays indicated that Cd treatment also activated the JAK3/STAT5 signaling to increase the expression of FasL, whereas Cd treatment reduced the expression of granzyme B in human mNK cells. Thus Cd treatment impaired the development of NK cells in the BM and bidirectionally regulated the cytotoxicity of mNK cells in the peripheral immune organs, which may extend our current understanding for the immunotoxicity of Cd.