High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon–liquid chromatography–tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0–1, 22.1–30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8–35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6–39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

The P2Y6-AMPK Pathway Triggers Autophagy during CSF-1-Induced Human Monocyte Differentiation and Is a Potential Target in CMML

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacological and genetic evidence indicate that autophagy plays pleiotropic functions in cellular homeostasis, development, survival and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by M-CSF. We have previously shown, using pharmacological inhibitors, siRNA approaches and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper CSF-1-driven differentiation of human and murine monocytes. We have also unraveled a role for autophagy in macrophage acquisition of phagocytic functions. Here, we determine that the CaMKKb-AMPKa1-ULK1 pathway is required for CSF-1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2Y6 to the induction of autophagy, and we decipher the signalling network that links the CSF-1 receptor to P2Y6-mediated autophagy and monocyte differentiation. Importantly, we show that the physiological P2Y6 ligand UDP and the specific P2Y6 agonist MRS2693 restore normal monocyte differentiation through re-induction of autophagy in primary myeloid cells from chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for P2Y6-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML. Disclosures No relevant conflicts of interest to declare.

Measures of immune recovery following autologous stem cell transplantation for multiple myeloma and outcome.

8581 Background: Autologous stem cell transplant (ASCT) improves survival in patients with multiple myeloma (MM). Recent studies have elucidated the relationship in ASCT between absolute lymphocyte count (ALC) recovery and improved survival, highlighting the importance of immune recovery. We conducted a retrospective analysis of patients with multiple myeloma to observe the impact of different measures of immune recovery at day 100 post-ASCT on outcome. Methods: Retrospective analysis of data from the existing clinical databases identified 1184 patients with multiple myeloma that underwent ASCT at Mayo Clinic between 1987-2011. Markers of immune recovery analyzed on day 100 status-post ASCT included ALC, monocyte count, and immunoglobulin levels. Kaplan Meier analysis was performed to determine progression free survival (PFS) and overall survival (OS). Results: Among the 1184 patients, median age at diagnosis of multiple myeloma was 57 (range, 23-75 yr), and median age at time of transplantation was 59 (range, 24-75 yr); 59% were male. The median OS and PFS post-transplant were 80 months and 34 months respectively. 62% of patients were alive 5-years post-transplant. The median OS was not improved with normal vs. abnormal IgG levels at day 100 (p=.298). In contrast, monocyte recovery was a significant predictor of OS. Patients with normal monocyte counts of greater than 0.3 x 10⁹ cells/L at day 100 showed superior survival (65 vs. 37 months, p= .001). Similarly, patients with normal monocyte recovery at day 15 post-transplant showed a significant survival benefit (68 vs. 50 months, p=.007). Early ALC recovery, which has been shown to be a positive prognostic indicator at day 15 post-transplant, was not prognostic at day 100 (p= .960). Conclusions: The present study further elucidates prognostic indicators after ASCT for MM highlighting the importance of various markers of immune recovery. Immunoglobulin recovery was not associated with superior survival, while monocyte recovery at day 15 and day 100 post-transplant translated to an improved survival and requires further study. It may be hypothesized that early immune system reconstitution may have a positive effect against disease post-transplant.

MNPs-Fe3o4 Induce Leukemia Cell Apoptosis by Oxidative Stress

Abstract 4304 The present study evaluated on the safe concentration for normal monocyte cells whether the magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) could induce caspase3-dependent apoptosis in SKM1 cells and U937 cells by oxidative damage. The average sizes of the particles were carefully determined by transmission electron microscopy. The viability of the cells after exposed to different concentration MNPs-Fe3O4 for 12h, 24h, 48h and 72h were detected by trypan blue staining. Cell cycle after exposure for 72h was analysed by propidium iodide (PI) staining. Apoptosis of MNPs-Fe3O4 group were detected and determined by Annexin V-FITC/PI double staining, 5,5’,6,6’-tetrachloro-1, 1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) probe and Wright - Giemsa staining. ROS in SKM1 cells and U937 cells after exposure were determined by flow cytometry after dichlorofluoroescein diacetate (DCFH-DA) staining. The changes of caspase3, survivin and bcl-rambo in the cells treatment with MNPs-Fe3O4 with or wtihout trolox for 48 hours were detected by western blot analysis. The significant decreased of cell viability caused by 100 μ M MNPS-Fe3O4 exposure were observed in SKM1 cells and u937 cells, not in normal monocytes cells (p < 0.05), the exposure also induced the SKM1 cells and u937 cells G0/G1 arrest (p < 0.05). Annexin V / PI staining assay show that 100 μ M MNPs-Fe3O4 group appeared more apoptosic rate in the U937 and SKM1 cells than the control group (p < 0.05), and on the same exposed concentration the apoptotic bodies could be frequently found in the U937 and SKM1 cells, while not in normal monocyte cells by Wright - Giemsa staining. The mitochondrial membrane potential in the two kinds of cells significantly decreased after exposed 100 μ M MNPs-Fe3O4 for 24h (p < 0.05), while pretreated with antioxidants trolox, the change was allevated (p < 0.05). DCFH-DA assay show that reactive oxygen species (ROS) generation increased in 6h of exposure in SKM1 cells and U937 cells (p < 0.05). Our results also showed the particle induced caspase3-dependent apoptosis in the SKM1 cells and U937 cells, while did not in normal monocyte cells, and increasing expression of bcl-rambo and decreasing expression of survivin involve in the process. These results suggest that on the safe concentration for normal monocyte cells MNPs-Fe3O4 could induce caspase3-dependent apoptosis in SKM1 cells and U937 cells by oxidative damage, moreover, increasing expression of bcl-rambo and decreasing expression of survivin involve in the process. Disclosures: No relevant conflicts of interest to declare.

Induction of interleukin-6 during human immunodeficiency virus infection

Interleukin-6 (IL-6), a multifunctional cytokine produced in monocytes, fibroblasts, and other cell types, is induced by a variety of stimuli, including bacteria, viruses, and other cytokines. When normal monocyte cultures were exposed to a monocytotropic strain of human immunodeficiency virus (HIV), HTLV-IIIBa-L, significant levels of IL-6 bioactivity were detected in the culture supernatants after 12 to 43 days of incubation, at a time when there was associated evidence of HIV production. Similarly, when normal monocyte cultures were cocultured with peripheral blood mononuclear cells from HIV-infected individuals, HIV replication in these cultures was associated with production of IL- 6. In further studies, we determined that mean serum levels of IL-6 bioactivity were abnormally elevated in HIV-seropositive individuals with stage 1/2 infection (25.2 x/divided by 1.8 U/mL) and stage 3/4 infection (46.1 x/divided by 1.7 U/mL) when compared with normals (1.6 x/divided by 1.2 U/mL). In contrast mean serum IL-6 levels were not different from normal in stage 5/6 infection (2.7 x/divided by 1.6 U/mL). A selected group of 12 HIV-seropositive individuals (stages 1, 2, and 3) who harbored HIV capable of replicating in T cells but not in monocyte cultures had a mean serum IL-6 level of 5.3 U/mL (x/divided by 1.5), a value significantly lower (P less than .004) than that measured in control HIV-seropositive individuals infected with monocytropic HIV (39 x/divided by 1.9 U/mL). In addition, serum IL-6 levels in HIV- seropositive individuals (stages 1 through 6) correlated directly with serum immunoglobulin G (IgG) levels (R = .74, P less than .001). Monocytes but not T cells are capable of a high level IL-6 production in vitro, and monocyte-derived IL-6 stimulates Ig production in activated B cells. Thus, HIV-seropositive individuals who often are infected with monocytotropic HIV and often display abnormally elevated serum IgG levels may exhibit these abnormalities as a consequence of abnormally elevated IL-6 levels induced by HIV.

Induction of interleukin-6 during human immunodeficiency virus infection

Interleukin-6 (IL-6), a multifunctional cytokine produced in monocytes, fibroblasts, and other cell types, is induced by a variety of stimuli, including bacteria, viruses, and other cytokines. When normal monocyte cultures were exposed to a monocytotropic strain of human immunodeficiency virus (HIV), HTLV-IIIBa-L, significant levels of IL-6 bioactivity were detected in the culture supernatants after 12 to 43 days of incubation, at a time when there was associated evidence of HIV production. Similarly, when normal monocyte cultures were cocultured with peripheral blood mononuclear cells from HIV-infected individuals, HIV replication in these cultures was associated with production of IL- 6. In further studies, we determined that mean serum levels of IL-6 bioactivity were abnormally elevated in HIV-seropositive individuals with stage 1/2 infection (25.2 x/divided by 1.8 U/mL) and stage 3/4 infection (46.1 x/divided by 1.7 U/mL) when compared with normals (1.6 x/divided by 1.2 U/mL). In contrast mean serum IL-6 levels were not different from normal in stage 5/6 infection (2.7 x/divided by 1.6 U/mL). A selected group of 12 HIV-seropositive individuals (stages 1, 2, and 3) who harbored HIV capable of replicating in T cells but not in monocyte cultures had a mean serum IL-6 level of 5.3 U/mL (x/divided by 1.5), a value significantly lower (P less than .004) than that measured in control HIV-seropositive individuals infected with monocytropic HIV (39 x/divided by 1.9 U/mL). In addition, serum IL-6 levels in HIV- seropositive individuals (stages 1 through 6) correlated directly with serum immunoglobulin G (IgG) levels (R = .74, P less than .001). Monocytes but not T cells are capable of a high level IL-6 production in vitro, and monocyte-derived IL-6 stimulates Ig production in activated B cells. Thus, HIV-seropositive individuals who often are infected with monocytotropic HIV and often display abnormally elevated serum IgG levels may exhibit these abnormalities as a consequence of abnormally elevated IL-6 levels induced by HIV.