Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Whole body deletion of Gpr68 does not change hematopoietic stem cell function.

G protein-coupled receptor 68 (GPR68) responds to extracellular protons, thus called the proton-sensing G protein-coupled receptor (GPCR), leading to activation of the phospholipase C-ß (PLCß)/calcium (Ca2+) pathway or the adenylyl cyclase (AC)/cyclic AMP (cAMP) pathway. We recently found that whole body deletion of Gpr68 (Gpr68-/- mice) reduced the number of B lymphocytes with age and during hematopoietic regeneration, such as in response to fluorouracil (5-FU) administration. This prompted us to characterize the hematopoietic stem cell (HSC) phenotype in Gpr68-/- mice. Despite high level of Gpr68 protein expression on HSC in bone marrow (BM), the pool size of HSC was unaltered in Gpr68-/- mice either under steady state or upon stress, including aging and 5-FU treatment. HSC from Gpr68-/- mice exhibited comparable cellular features, such as cell cycle quiescence and cell survival. HSC from Gpr68-/- mice also exhibited comparable competitiveness after serial transplantation. Surprisingly, cytosolic Ca2+ accumulation was increased in HSC from Gpr68-/- mice. In contrast, cAMP levels were reduced in hematopoietic stem and progenitor cells (HSPC) from Gpr68-/- mice. Intriguingly, we found high level of Gpr68 protein expression on non-hematopoietic cells in BM, especially endothelial cells that function as HSC niche. In addition, expression of other proton-sensing GPCR was upregulated in HSPC from Gpr68-/- mice. Our studies suggest that Gpr68-/- mice display insignificant phenotype on HSC biology, possibly due to the function of Gpr68 in non-hematopoietic cells and/or the compensatory effects from other proton-sensing GPCR.

Cyclosporine enhances the sensitivity to lenalidomide in MDS/AML in vitro.

Our previous study revealed that expression of G protein-coupled receptor 68 (GPR68) was upregulated in MDSL cells, a cell line representing myelodysplastic syndromes (MDS), in response to lenalidomide (LEN), and mediated a calcium/calpain proapoptotic pathway. Isx, a GPR68 agonist, enhanced the sensitivity to LEN in MDSL cells. The fact that Isx is not a U.S. Food and Drug Administration-approved drug prompts us to look for alternative candidates that could enhance the sensitivity of LEN in MDS as well as other hematologic malignancies, such as acute myeloid leukemia (AML). In the study described here, we found that regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of calcineurin (CaN), was upregulated in MDSL cells in response to LEN, possibly through degradation of IKZF1. Consistently, cyclosporin (Cys), a pharmacological inhibitor of CaN, inhibited the activity of CaN and induced apoptosis in MDSL cells, indicating that CaN provided a prosurvival signal in MDSL cells. In addition, Cys enhanced the cytotoxic effect of LEN in MDS/AML cell lines as well as primary bone marrow cells from MDS patients and AML patient-derived xenograft models. Intriguingly, pretreatment with LEN reversed the suppressive effect of Cys on T-cell activation. Our study suggests a novel mechanism of action of LEN in mediating cytotoxicity in MDS/AML via upregulation of RCAN1, thus inhibiting the CaN prosurvival pathway. Our study also suggests that Cys enhances the sensitivity to LEN in MDS/AML cells without compromising T-cell activation.

G protein-coupled receptor 68 increases the number of B lymphocytes.

G protein-coupled receptor 68 (GPR68) is a proton sensor that is activated upon binding to extracellular protons. We have previously found that GPR68 induces a proapoptotic pathway in bone marrow (BM) cells from the patients with myelodysplastic syndromes (MDS) after treated with lenalidomide. However, the function of GPR68 in normal hematopoietic cells remains unclear. With genetic loss of function approach, we found reduced frequency and number of B lymphocytes in the peripheral blood (PB) of whole body Gpr68-/- mice compared to control littermates upon aging. During hematopoietic regeneration, such as in response to fluorouracil (5-FU), we also found reduced frequency and number of B lymphocytes in Gpr68-/- mice compared to wild type mice. Mechanism studies revealed that Gpr68 expression was upregulated in B lymphocytes of BM during aging and in hematopoietic progenitor cells after treatment with 5-FU. In addition, activation of Gpr68 by its activators increased the frequency and number of B lymphocytes. Our studies indicate that Gpr68 expression is upregulated in hematopoietic cells upon aging and during hematopoietic regeneration that ends up with increased number of B lymphocytes.