Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

ObjectivesCell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE).MethodsEvaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR.ResultsElevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores.ConclusionAlterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE

Polyploid Adipose Stem Cells Shift the Balance of IGF1/IGFBP2 to Promote the Growth of Breast Cancer

Background: The close proximity of adipose tissue and mammary epithelium predispose involvement of adipose cells in breast cancer development. Adipose-tissue stem cells (ASCs) contribute to tumor stroma and promote growth of cancer cells. In our previous study, we have shown that murine ASCs, which undergo polyploidization during their prolonged in vitro culturing, enhanced the proliferation of 4T1 murine breast cancer cells in IGF1 dependent manner. Aims: In the present study, our aim was to clarify the regulation of ASC-derived IGF1. Methods: 4T1 murine breast carcinoma cells were co-transplanted with visceral fat-derived ASCs (vASC) or with the polyploid ASC.B6 cell line into female BALB/c mice and tumor growth and lung metastasis were monitored. The conditioned media of vASCs and ASC.B6 cells were subjected to LC-MS/MS analysis and the production of IGFBP2 was verified by Western blotting. The regulatory effect was examined by adding recombinant IGFBP2 to the co-culture of ASC.B6 and 4T1. Akt/protein kinase B (PKB) activation was detected by Western blotting. Results: Polyploid ASCs promoted the tumor growth and metastasis more potently than vASCs with normal karyotype. vASCs produced the IGF1 regulator IGFBP2, which inhibited proliferation of 4T1 cells. Downregulation of IGFBP2 by polyploidization of ASCs and enhanced secretion of IGF1 allowed survival signaling in 4T1 cells, leading to Akt phosphorylation. Conclusions: Our results implicate that ASCs in the tumor microenvironment actively regulate the growth of breast cancer cells through the IGF/IGFBP system

Synthesis of N-peptide-6-amino-D-luciferin Conjugates

A general strategy for the synthesis of N-peptide-6-amino-D-luciferin conjugates has been developed. The applicability of the strategy was demonstrated with the preparation of a known substrate, N-Z-Asp-Glu-Val-Asp-6-amino-D-luciferin (N-Z-DEVD-aLuc). N-Z-DEVD-aLuc was obtained via a hybrid liquid/solid phase synthesis method, in which the appropriately protected C-terminal amino acid was coupled to 6-amino-2-cyanobenzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was then attached to resin. The resulting loaded resin was used for the solid-phase synthesis of the desired N-peptide-6-amino-D-luciferin conjugate without difficulties, which was then attested with NMR spectroscopy and LC-MS, and successfully tested in a bioluminescent system

Multi-Dimensional Immuno-Profiling of Drosophila Hemocytes by Single Cell Mass Cytometry

Single cell mass cytometry (SCMC) combines features of traditional flow cytometry (FACS) with mass spectrometry and allows the measurement of several parameters at the single cell level, thus permitting a complex analysis of biological regulatory mechanisms. We optimized this platform to analyze the cellular elements, the hemocytes, of the Drosophila innate immune system. We have metal-conjugated six antibodies against cell surface antigens (H2, H3, H18, L1, L4, P1), against two intracellular antigens (3A5, L2) and one anti-IgM for the detection of L6 surface antigen, as well as one anti-GFP for the detection of crystal cells in the immune induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive, in immune induced states, in tumorous mutants (hopTum bearing a driver mutation and l(3)mbn1 carrying deficiency of a tumor suppressor) as well as in stem cell maintenance defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets, lamellocytes, plasmatocytes, crystal cell, and delineated the unique immunophenotype of the mutants. We have identified sub-populations of L2+/P1+ (l(3)mbn1), L2+/L4+/P1+ (hopTum) transitional phenotype cells in the tumorous strains and a sub-population of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time, that mass cytometry, a recent single cell technology combined with multidimensional bioinformatic analysis represents a versatile and powerful tool to deeply analyze at protein level the regulation of cell mediated immunity of Drosophila

Investigation of Anticancer Properties of Monoterpene-Aminopyrimidine Hybrids on A2780 Ovarian Cancer Cells

The present study aimed to characterize the antiproliferative and antimetastatic properties of two recently synthesized monoterpene-aminopyrimidine hybrids (1 and 2) on A2780 ovary cancer cells. Both agents exerted a more pronounced cell growth inhibitory action than the reference agent cisplatin, as determined by the MTT assay. Tumor selectivity was assessed using non-cancerous fibroblast cells. Hybrids 1 and 2 induced changes in cell morphology and membrane integrity in A2780 cells, as evidenced by Hoechst 33258–propidium iodide fluorescent staining. Cell cycle analysis by flow cytometry revealed substantial changes in the distribution of A2780 ovarian cancer cells, with an increased rate in the subG1 and G2/M phases, at the expense of the G1 cell population. Moreover, the tested molecules accelerated tubulin polymerization in a cell-free in vitro system. The antimetastatic properties of both tested compounds were investigated by wound healing and Boyden chamber assays after 24 and 48 h of incubation. Treatment with 1 and 2 resulted in time- and concentration-dependent inhibition of migration and invasion of A2780 cancer cells. These results support that the tested agents may be worth of further investigation as promising anticancer drug candidates

SMALL MOLECULES DU192, DU283 AND DU325 INDUCE DIFFERENTIATION AND APOPTOSIS OF HUMAN ACUTE PROMYELOCYTIC LEUKEMIA CELLS

Acute myelogenous leukemia (AML) originates from myeloid stem cells or myeloid blasts halted in an immature state during haematopoiesis. AML represents a group of heterogeneous forms of myeloid malignancies with diverse genetic abnormalities and different stages of myeloid differentiation. The human cell line, HL-60 used in this study belongs to a sub-type of AML, namely acute promyelocytic leukemia (APL). Another pathologic condition of myeloid expansion is the ”emergency” granulo-monocytopoiesis in most of the solid malignancies in which, an army of immature myeloid cells leave the bone marrow, called monocytic and granulocytic myeloid-derived suppressor cells (MDSCs). In contrast to AML, MDSCs are not malignant cells, but promote angiogensesis and immunosuppression leading to the progression of cancer. Both in AML and in solid malignancies the differentiation of immature myeloid cells is an already established therapeutic concept. Since the differentiation of AML cells is frequently followed by apoptosis or increases the sensitivity to chemotherapy, we have screened a library of small molecules to mature the human prototype cells, HL-60. In the resazurin assay small molecules DU192, DU283 and DU325 confounded viability of HL-60 cells, half-inhibitory concentration (IC50) values were as follows: 940 nM, 210 nM and 20 nM, respectively. IC50 could not be determined for human primary fibroblasts in the applied concentration range (1.6 nM - 5 µM). Using flow cytometry we obtained ERK phosphorylation as an early response to DU325 stimulation followed by the increase of the percentage of the Bcl-xl and pAkt bright cells. The expression of the members of the AP-1 TF complex, a driver of cellular differentiation, c-Fos, JunB, c-Jun and JunD were elevated on a concentration and time dependent manner detected by qRT-PCR. As a proof of cellular differentiation the expression of haematopoietic stem cell markers CD33 and CD34 decreased. Due to maturation the size and granularity of HL-60 cells increased upon treatment. Matured myeloid cell marker CD11b elevated on the cell surface detected by flow cytometry. We confirmed that differentiation of HL-60 cells was accompanied by apoptosis. We could detect AnnexinV+/PI- early and AnnexinV+/PI+ late apoptotic populations after 24h of treatment. Caspase-3 activated gradually by time detected by the percentage of active caspase-3 positive cells by flow cytometry and the digestion of zDEVD – amino-Luciferin. Finally, as a proof of massive cell death, we have shown the appearance of the hypo-diploid apoptotic cells in the sub-G1 population and the leakage of the lactate-dehydrogenase into the supernatant. We conclude that DU molecules differentiated immature HL-60 cells, which was followed by apoptosis. We propose to further investigate the effects of DU325 on additional human AML cells obtained from clinical samples. On the other hand we plan to systematically investigate the effect of DU325 on the expansion of immature MDSCs in solid malignancies. Funding: GINOP-2.3.2-15-2016-00030 (LGP); János Bolyai Research Scholarship (GJSz, BO/00139/17/8

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer

Enantioselective Synthesis of 8-Hydroxyquinoline Derivative, Q134 as a Hypoxic Adaptation Inducing Agent

Hypoxia is a common feature of neurodegenerative diseases, including Alzheimer’s disease that may be responsible for disease pathogenesis and progression. Therefore, the hypoxia-inducible factor (HIF)1 system, responsible for hypoxic adaptation, is a potential therapeutic target to combat these diseases by activators of cytoprotective protein induction. We have selected a candidate molecule from our cytoprotective hydroxyquinoline library and developed a novel enantioselective synthesis for the production of its enantiomers. The use of quinidine or quinine as a catalyst enabled the preparation of enantiomer-pure products. We have utilized in vitro assays to evaluate cytoprotective activity, a fluorescence-activated cell sorting (FACS) based assay measuring mitochondrial membrane potential changes, and gene and protein expression analysis. Our data showed that the enantiomers of Q134 showed potent and similar activity in all tested assays. We have concluded that the enantiomers exert their cytoprotective activity via the HIF1 system through HIF1A protein stabilization

Role of IL-24 in the mucosal remodeling of children with coeliac disease

Background Recently, involvement of IL-19, IL-20 and IL-24 has been reported in inflammatory diseases associated with tissue remodeling. However, their impact on the pathomechanism of coeliac disease (CD) is still completely unknown. Methods Expression of IL19, IL20 and IL24 was measured by real-time RT-PCR, protein amount of IL-24, α smooth muscle actin (α-SMA) and fibronectin (FN) was determined by Western-blot analysis in the duodenal biopsies of therapy naive children with CD and controls. Localization of IL-24 and IL-20RB was investigated by immunofluorescent staining in the duodenal mucosa. Effect of recombinant IL-1β, TNF-α, TGF-β and IL-17 treatment on the expression of IL19, IL20, IL24 and their receptors was investigated by real-time RT-PCR in small intestinal epithelial cells (FHs74Int), in primary duodenal myofibroblasts (pdMFs) and in peripheral blood mononuclear cells (PBMCs). Effect of IL-24 on H2O2 treated FHs74Int cells and on pdMFs was measured by MTT, LDH, Annexin V assays, real-time RT-PCR and by fluorescent microscopy. Results We found increased level of IL-24 (3.3×, p < 0.05), α-SMA (2.4×, p < 0.05) and FN (2.3×, p < 0.05) in the duodenal mucosa and increased expression of IL19 (3.6×, p < 0.05) and IL24 (5.2×, p < 0.05) in the PBMCs of children with CD compared to that of controls. IL-1β was a strong inducer of IL24 expression of FHs74Int cells (9.9×, p < 0.05), pdMFs (552.9×, p < 0.05) or PBMCs (17.2×, p < 0.05), as well. IL-24 treatment reduced the number of apoptotic cells (0.5×, p < 0.05) and decreased the expression of inflammatory factors, including IL1A, IL6 and TNF of H2O2-treated FHs74Int cells. IL-24 decreased the proliferation (0.6×, p < 0.05) of PDGF-B treated pdMFs. Moreover, IL-24 treatment altered the morphology of pdMFs by influencing the size of the angles between stress fibers and the longitudinal axis of the cells (2.0×, p < 0.05) and the expression of cytoskeletal components, including ACTA2, ACTB, VIM, SNAI1 and SNAI2. Conclusion Our results suggest that IL-24 plays a significant role in the maintenance of duodenal mucosal integrity in CD

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer