Analysis of PD-1 expression in the monocyte subsets from non-septic and septic preterm neonates

Programmed death-1 (PD-1) receptor system represents a part of recently reported immunoregulatory pathway. PD-1 is an immune checkpoint molecule, which plays an important role in downregulating the immune system proinflammatory activity. Until recently, PD-1 expression was not established on immune cells of the preterm infants. The study objectives were to confirm expression of the PD-1 receptors on the monocytes isolated from very low birth weight newborns (VLBW), and to analyze their expression during the first week of life and late-onset sepsis. Peripheral blood mononuclear cells were isolated from 76 VLBW patients without early-onset sepsis on their 5th day of life (DOL). PD-1 expression was determined on the monocyte subsets (classical, intermediate, non-classical) by flow cytometry. In case of late-onset sepsis (LOS), the same analysis was performed. Our results demonstrated that on the 5th DOL, PD-1 receptors were present in all the monocyte subsets. Children, whose mothers had received antenatal steroids, presented higher absolute numbers of non-classical monocytes with PD-1 expression. Infants born extremely preterm who later developed LOS, initially showed a lower percentage of PD-1 receptor-positive intermediate monocytes in comparison to neonates born very preterm. During LOS, we observed a rise in the percentage of classical monocytes with PD-1 expression. In case of septic shock or fatal outcome, there was a higher percentage and absolute count of intermediate monocytes with PD-1 expression in comparison to children without these complications. In conclusion, monocytes from VLBW children express PD-1 receptors. Antenatal steroid administration seems to induce PD-1 receptor expression in the non-classical monocytes. PD-1 might play a role in immunosuppressive phase of sepsis in the prematurely born children with septic shock and fatal outcome

A novel high-content immunofluorescence assay as a tool to identify at the single cell level γ-globin inducing compounds

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ-and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathie

Analysis of the monocyte subpopulations in the patients with or without septic shock.

<p>(A) Absolute numbers of monocyte subsets. (B) Percentages of PD-1 positive cells. (C) Absolute numbers of PD-1 positive cells. Data presented as median and IQR (box), compared with Wilcoxon test. Whiskers—range within 1.5 IQR. Classical monocytes are presented as a black graph, intermediate monocytes are presented as a graph with horizontal lines, whereas non-classical monocytes are presented as a graph with vertical lines. P-value was significant in case of *p<0.05.</p

Comparison of selected demographic variables and hospitalization data of the patients in the two studied groups.

<p>Comparison of selected demographic variables and hospitalization data of the patients in the two studied groups.</p

Monocyte populations and PD-1 expression in the before-LOS group of VLBW infants.

<p>Patients were subdivided into groups based on the level of their gestational age at birth. (A) Absolute numbers of monocyte subsets. (B) Percentages of PD-1 expressing monocytes. (C) Absolute numbers of PD-1 expressing monocytes. Data presented as median and IQR (box), compared with Wilcoxon test. Whiskers—range within 1.5 IQR. Classical monocytes are presented as a black graph, intermediate monocytes are presented as a graph with horizontal lines, whereas non-classical monocytes are presented as a graph with vertical lines. P-value was significant in case of *p<0.05.</p

Counts of monocyte subsets, monocyte subsets with PD-1 expression, and a percentage of PD-1⁺ cells within each monocyte subset in the blood samples from VLBW infants collected on the 5^th DOL.

<p>Data presented as median and IQR.</p

Analysis of the monocyte subpopulations in VLBW infants who either did nor did not survived LOS.

<p>(A) Absolute numbers of monocyte subsets. (B) Percentages of PD-1-positive cells. (C) Absolute numbers of PD-1 positive cells. Data presented as median and IQR (box), compared with Wilcoxon test. Whiskers—range within 1.5 IQR. Classical monocytes are presented as a black graph, intermediate monocytes are presented as a graph with horizontal lines, whereas non-classical monocytes are presented as a graph with vertical lines. P-value was significant in case of *p<0.05.</p

High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.

<p>A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and dacinostat (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s003" target="_blank">S3 Fig</a>). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>.</p

High-content analysis of compound-induced changes in globins accumulation.

<p>β-K562 cells were treated with 800μM hydroxyurea and 900μM butyric acid (n = 3, a representative experiment is shown here) and the same cells were analyzed in parallel by immunofluorescence and by RTqPCR 4 days after the addition of the drugs. A) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>. C) RTqPCR on α-, γ- and β- globins. Histograms show the relative levels of expression relative to GAPDH. D) Confocal analysis of β-K562 cells untreated or treated with HU as in panel A and subjected to a quadruple staining with Hoechst (blue), anti β- (green), anti γ-globin (red) and anti-CD235a (white). Magnification: 20x. Right panel: 40x magnification of individual cells γ⁺CD235a⁺ or β⁺CD235a⁺ double positive and γ⁺β⁺CD235a⁺ triple positive, respectively.</p

Analysis of γ/β globin levels by immunofluorescence and automated image capture.

<p>A) Image acquisition and analysis for β-K562 and K562. Merged signals of DNA (Hoechst-33342), β-globin and γ-globin are read in channel 1 (Ch1), channel 2 (Ch2) and channel 3 (Ch3), respectively (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s001" target="_blank">S1D Fig</a>). Bar = 50μm. The intensity value of signals is automatically assigned by the instrument and converted into a corresponding intensity of color. The relative scatter plots show the distribution of double γ^-β^- negative, single γ⁺β^- positive, single γ^-β⁺ positive and double γ⁺β⁺ positive cells (x axis: FITC-β-globin; y axis: PE-γ-globin). Numbers within plots refer to the averaged percentage of cells within each population from three independent experiments (n = 3). The relative st.errors are shown in panel C: *p<0,05; ** p<0,01; ***p<0,001. B) Quantitative fluorescence imaging of single cells: cells numbered from 1 to 6 in panel A are taken as an example of γ^-β^- double negative (1 and 2), single γ⁺β^- positive (5), single γ^-β⁺ positive (4) and γ⁺β⁺ double positive (3 and 6). C) Statistical analysis (n = 3): γ^-β^- cells; red: γ⁺β^- cells; yellow: γ⁺β⁺ cells; green: γ^-β⁺ cells. D) RTqPCR on α, ε, γ- and β-globins. Histograms show the relative levels of expression normalized on glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n≥3, statistical analysis: *p<0,05; **p<0,01; ***p<0,001.</p