Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex

Summary: Here, we present a protocol for differential multi-omic analyses of distinct cell types in the developing mouse cerebral cortex. We describe steps for in utero electroporation, subsequent flow-cytometry-based isolation of developing mouse cortical cells, bulk RNA sequencing or quantitative liquid chromatography-tandem mass spectrometry, and bioinformatic analyses. This protocol can be applied to compare the proteomes and transcriptomes of developing mouse cortical cell populations after various manipulations (e.g., epigenetic).For complete details on the use and execution of this protocol, please refer to Meka et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

<div><p>Background</p><p>Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis.</p><p>Methods</p><p>Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following <i>ex vivo</i> stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression.</p><p>Results</p><p>Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman’s rho -0.59, P<0.001)</p><p>Conclusion</p><p>In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness.</p></div

Low expression of human leucocyte antigen receptor alpha (HLA-DRA) is associated with unresponsiveness of whole blood to lipopolysaccharide (LPS) and is associated with disease severity.

<p>(A) Whole blood was stimulated with 500 pg/mL LPS for 3h and TNFα levels were determined in the supernatant. The x-axis shows fold changes from baseline TNFα levels after <i>ex vivo</i> stimulation. The y-axis shows HLA-DRA expression levels assessed by quantitative polymerase chain reaction (qPCR) using the delta-delta Ct method. Linear regression analysis was performed after transformation to a logarithmic scale to reach normal distribution. Solid line: regression for patients; dotted line: regression for all study participants. Circles represent data from patients and dots represent data from controls. (B) TNFα:HLA-DRA ratio correlates with the SOFA score. Low TNFα response to LPS stimulation is associated with low expression levels of HLA-DRA as shown in (A). The ratio TNFα:HLA-DRA correlates with severity of the disease as measured by the SOFA score. Spearman’s rank test was used to calculate correlation. Circles represent ratios from individual patients.</p

Characteristics of study groups.

<p>Characteristics of study groups.</p

Monocytosis but loss of protein surface and gene expression of human leucocyte antigen DR (HLA-DR) is observed in sepsis patients.

<p>(A) Number of monocytes in cells/mL shows significant higher numbers of monocytes/mL in sepsis patients (n = 9) compared to controls (n = 10). (B) Protein surface expression levels of HLA-DR by monocytes are shown as a representative histogram and (C) with the corresponding analysis of median fluorescence intensity (MFI). Sepsis patients show reduced HLA-DR surface expression compared to controls. (A-C) Whole blood was stained for cell surface molecules (CD14 and HLA-DR) and analyzed by flow cytometry (FACS). Data are presented as box and whisker plots with median and interquartile range and statistical analysis was performed using non-parametric Mann-Whitney-U test. (D) Gene expression of HLA-DRA is reduced in sepsis patients. mRNA was prepared from whole blood and HLA-DRA expression levels were assessed by quantitative PCR (qPCR). HLA-DRA expression levels are normalized to internal control gene peptidylpropylisomerase B (PPIB). Data are presented using the delta-delta Ct method and as box and whisker plots showing the median with interquartile range. Statistical analysis was performed using non-parametric Mann-Whitney-U test. (E) Spearman’s rank correlation is presented with median fluorescence intensity (MFI) for HLA-DR protein on the x-axis and mRNA expression of HLA-DR presented as delta-delta Ct on the y-axis. Each circle represents a data set from an individual patient. CD: cluster of differentiation, mRNA: messenger RNA, FMO: Fluorescence Minus One controls, PCR: polymerase chain reaction. P<0.05:*; P<0.01:**; P<0.001:***.</p

Innate response of CD39^-/- mice against <i>L</i>. <i>monocytogenes</i>.

<p>Wildtype and CD39^-/- mice were i.v. infected with 5×10³ Lm. (A) Bacterial burden in spleens was determined at d2 post infection. Combined results for 10 individually analyzed mice from two independent experiments and the median are shown. cfu, colony forming units. Mann Whitney test, * p<0.05. (B) Wildtype mice were infected with 1×10⁵ LmOVA. At the indicated time points, neutrophils and inflammatory monocytes from the spleen were analyzed for the expression of CD39 and CD73 by flow cytometry. Representative concatenated dot plots for surface expression of CD39 and CD73 on neutrophils and inflammatory monocytes are shown. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197151#pone.0197151.s002" target="_blank">S2 Fig</a> for mean values for groups of mice.) (C, D) Spleen cells from wildtype and CD39^-/- mice were cultured with Lm overnight. Supernatants were collected and the concentration of IL-1β (C) and TNF-α (D) was determined by ELISA. Bars present the mean ± SEM of eight values. (E) Wildtype and CD39^-/- mice were i.v. infected with 5×10³ Lm. On day 2 post infection, spleen cells were isolated and expression of TNF-α was directly analyzed in inflammatory monocytes by intracellular cytokine staining and flow cytometry. Representative histograms and frequency of TNF-α⁺ inflammatory monocytes are given. Bars present the mean ± SEM of five individually analyzed mice. Results are representative for two independent experiments. Unpaired t test, ** p<0.01.</p

CD39 and CD73 expression profiles of human peripheral blood T-cell subsets.

<p>(A) Gating strategy for conventional CD4⁺ and CD8⁺ T cells from human peripheral blood. CD25⁺CD127^— T_reg cells were excluded. (B) CD39 and CD73 as well as CCR7 and CD45RA expression profile of conventional CD4⁺ and CD8⁺ T cells. Naive: CCR7⁺CD45RA⁺, effector/effector memory (EM): CCR7^—CD45RA^—, central memory (CM): CCR7⁺CD45RA^— and TEMRA: CCR7^—CD45RA⁺. (C) Percentages of CD39 and CD73 expression on CD4⁺ and CD8⁺ T-cell subsets from the blood of 10 healthy donors and the means are shown. ANOVA multiple comparison, * p<0.05, ** p<0.01, *** p<0.001.</p

CD39 and CD73 expression on CD4⁺ and CD8⁺ T cells from the synovial fluid of inflamed joints.

<p>T cells from the peripheral blood and synovial fluid of JIA patients were analyzed for the expression of HLA-DR, CD39 and CD73. T_reg cells were excluded from analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197151#pone.0197151.g002" target="_blank">Fig 2A</a> for gating strategy). (A, B) Representative expression profiles for CD39, CD73 and HLA-DR on conventional CD4⁺ T cells (A) and CD8⁺ T cells (B) from peripheral blood (PB) and synovial fluid (SF). (C) Percentages of HLA-DR⁺ cells among CD4⁺ and CD8⁺ T cells in PB and SF. (D) Percentages of CD39⁺ cells (top) and of CD73⁺ cells (bottom) among HLA-DR⁺ and HLA-DR^—subsets of CD4⁺ and CD8⁺ T cells in peripheral PB and SF. Results for blood and SF of 6 JIA patients are shown. Paired t test, * p<0.05, ** p<0.01, *** p<0.001, ns p>0.05.</p