Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is... Show moreMass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations. Show less
Context: Adult obesity is associated with chronic low-grade inflammation and may give rise to future chronic disease. However, it is unclear whether adiposity-related inflammation is already... Show moreContext: Adult obesity is associated with chronic low-grade inflammation and may give rise to future chronic disease. However, it is unclear whether adiposity-related inflammation is already apparent in childhood.Objective: To study associations between child adiposity measures with circulating monocytes and naive and memory subsets in CD4, CD8, and gamma delta T cell lineages.Methods: Ten-year-old children (n = 890) from the Generation R Cohort underwent dual-energy x-ray absorptiometry and magnetic resonance imaging for body composition (body mass index [BMI], fat mass index [FMI], android-to-gynoid fat mass ratio, visceral fat index, liver fat fraction). Blood samples were taken for detailed immunophenotyping of leukocytes by 11-color flow cytometry.Results: Several statistically significant associations were observed. A 1-SD increase in total FMI was associated with +8.4% (95% CI 2.0, 15.2) V delta 2(+)V gamma 9(+) and +7.4% (95% CI 2.4, 12.5) CD8(TEMRO)(+) cell numbers. A 1-SD increase in visceral fat index was associated with +10.7% (95% CI 3.3, 18.7) V delta 2(+)V gamma 9(+) and +8.3% (95% CI 2.6, 14.4) CD8(TEMRO)(+) cell numbers. Higher android-to-gynoid fat mass ratio was only associated with higher V delta 2(+)V gamma 9(+) T cells. Liver fat was associated with higher CD8(TEMRO)(+) cells but not with V delta 2(+)V gamma 9(+) T cells. Only liver fat was associated with lower Th17 cell numbers: a 1-SD increase was associated with -8.9% (95% CI -13.7, -3.7) Th17 cells. No associations for total CD8(+), CD4(+) T cells, or monocytes were observed. BMI was not associated with immune cells.Conclusion: Higher V delta 2(+)V gamma 9(+) and CD8(TEMRO)(+) cell numbers in children with higher visceral fat index could reflect presence of adiposity-related inflammation in children with adiposity of a general population. Show less
Oosenbrug, T.; Graaff, M.J. van de; Haks, M.C.; Kasteren, S. van; Ressing, M.E. 2020
Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines,... Show moreSurface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines, including type I interferons (IFN-I). Downstream of TLR4, IFN beta secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear whether this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-kappa B, driven by the kinases TBK1 and TAK1-IKK beta, respectively. TLR2-stimulated monocytes produced modest IFN beta levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses. Show less
Objective: Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether... Show moreObjective: Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether Netrin-1 is associated with different stages of atherosclerosis. Approach and Results: Plasma Netrin-1 levels were measured in different patient cohorts: (1) 22 patients with high cardiovascular risk who underwent arterial wall inflammation assessment using positron-emission tomography / computed tomography, (2) 168 patients with a positive family history of premature atherosclerosis in whom coronary artery calcium scores were obtained, and (3) 104 patients with chest pain who underwent coronary computed tomography angiography imaging to evaluate plaque vulnerability and burden. Netrin-1 plasma levels were negatively correlated with arterial wall inflammation (beta, -0.01 [95% CI, 0.02 to -0.01] R-2, 0.61; P<0.0001), and concentrations of Netrin-1 were significantly lower when atherosclerosis was present compared with individuals without atherosclerosis (28.01 versus 10.51 ng/mL, P<0.001). There was no difference in Netrin-1 plasma concentrations between patients with stable versus unstable plaques (11.17 versus 11.74 ng/mL, P=0.511). However, Netrin-1 plasma levels were negatively correlated to total plaque volume (beta, -0.09 [95% CI, -0.11 to -0.08] R-2, 0.57, P<0.0001), calcified plaque volumes (beta, -0.10 [95% CI, -0.12 to -0.08] R-2, 0.53; P<0.0001), and noncalcified plaque volumes (beta, -0.08 [95% CI, -0.10 to -0.06] R-2, 0.41; P<0.0001). Treatment of inflammatory stimulated endothelial cells with plasma with high Netrin-1 level resulted in reduced endothelial inflammation and consequently, less monocyte adhesion. ConclusionS: Netrin-1 plasma levels are lower in patients with subclinical atherosclerosis and in patients with arterial wall inflammation. Netrin-1 is not associated with plaque vulnerability; however, it is negatively correlated to plaque burden, suggesting that Netrin-1 is involved in some, but not all, stages of atherosclerosis. Show less
