Multiple linear regression models for the association between annual change in MMSE score during follow-up and baseline levels of CSF CCL2, including potential confounding factors.

<p>Values are given as standardized beta coefficients (t-values).</p><p>*P<0.05;</p><p>**P<0.01.</p

The baseline levels of CCL2 in the cerebrospinal fluid correlated positively with the annual change in MMSE score during clinical follow-up (<i>r</i>_s = 0.42, p = 0.004).

<p>The baseline levels of CCL2 in the cerebrospinal fluid correlated positively with the annual change in MMSE score during clinical follow-up (<i>r</i>_s = 0.42, p = 0.004).</p

The levels of chemokines in cerebrospinal fluid (CSF) and plasma obtained at baseline.

<p>The chemokine levels are given in pg/ml. Only <i>P</i>-values<0.01 are considered significant, because of correction for multiple comparisons (all groups were compared to both controls and stable MCI).</p>a<p><i>P</i><0.01 vs Controls.</p>b<p><i>P</i><0.01 vs Stable MCI.</p><p>Abbreviations: Stable MCI, patients with MCI with stable cognitive functions during a follow-up period of 5.2 years; MCI-AD, patients with MCI who developed Alzheimer's disease during follow-up; MCI-other, patients with MCI who developed other types of dementia during follow-up; CSF, cerebrospinal fluid.</p

Demographic data of included subjects.

<p>Data are the mean (± standard deviation) or number (%).Only <i>P</i>-values<0.01 are considered significant, because of correction for multiple comparisons (all groups were compared to both controls and stable MCI).</p>a<p><i>P</i><0.01 vs Controls.</p>b<p><i>P</i><0.01 vs Stable MCI.</p><p>Abbreviations: Stable MCI, patients with MCI with stable cognitive functions during a follow-up period of 5.2 years; MCI-AD, patients with MCI who developed Alzheimer's disease during follow-up; MCI-other, patients with MCI who developed other types of dementia during follow-up; CSF, cerebrospinal fluid; <i>APOE</i>, apolipoprotein E; MMSE, Mini-Mental State Examination.</p

The clinical value of combining baseline levels of CSF CCL2 with standards CSF biomarkers (i.e. Tau, P-tau and Aβ42) in the whole MCI cohort.

<p>MCI patients with an AD-indicative CSF biomarker pattern (Tau>350 pg/ml and Aβ42/P-tau ratio <6.5; red squares) declined cognitively during follow-up, because many of these patients developed AD dementia, which was not the case for those with a normal CSF biomarker pattern (green circles). Importantly, the disease progression rate was significantly higher in MCI patients with an AD-indicative CSF biomarker pattern, who also had high CCL2 levels (in the third tertile) when compared to those who exhibited lower levels (in the first tertile) (p<0.05).</p

Genes of interest investigated in the three monocyte subsets.

<p>Protein function adapted from <a href="http://GeneCards.org" target="_blank">GeneCards.org</a></p><p>Genes of interest investigated in the three monocyte subsets.</p

Multimodal variation in expression levels across the three monocyte subsets.

<p>Violin plot demonstrating multimodal variation in gene expression levels of the 85 genes examined in the monocyte subsets. The classical monocytes, intermediate monocytes and non-classical monocytes are indicated in the figure by red, green and blue, respectively. The data depict the multimodal expression levels of the genes listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144351#pone.0144351.t004" target="_blank">Table 4</a> calculated by using the Hartigans dip test (<i>P <</i> 0.05).</p

Expression level of genes deviating in identified subgroups.

<p>Subgroups of cells were identified based on the PCA of single-cell PCR gene expression analysis data. One subgroup among the classical monocytes, intermediate monocytes and non-classical monocytes, and co-expression of genes within the subgroups were assessed using a student T test (<i>P <</i> 0.05). A) Bar graph demonstrating the differentially expressed genes by the subgroup within the classical monocyte subset identified on the PCA score plot. The subgroup is marked by filled red circles in the PCA score plot. B) Bar graph demonstrating the differentially expressed genes by the subgroup within the intermediate monocyte subset identified on the PCA score plot. The subgroup is marked by filled green triangles in the PCA score plot. C) Bar graph demonstrating the differentially expressed genes by the subgroup within the non-classical monocyte subset identified on the PCA score plot. The subgroup is marked by filled blue pluses in the PCA score plot.</p

Relative Log2 transformed gene expression levels of the three subsets and statistical significance the subsets in between.

<p>C = Classical, I = Intermediate, NC = Non-Classical</p><p>Relative Log2 transformed gene expression levels of the three subsets and statistical significance the subsets in between.</p

Single-cell gene expression analysis on human monocytes.

<p>Human monocytes were single-cell sorted according to the expression of the cell surface markers CD14 and CD16 and gene expression on single cells was assessed. A) Representative plot of flow cytometry analysis demonstrating the monocyte subset gating strategy, used to single-cell sort monocytes from the three classified monocyte subsets (<i>n =</i> 1). Classical (CD14⁺⁺CD16^-) monocytes, intermediate (CD14⁺⁺CD16⁺) monocytes and non-classical (CD14⁺⁺CD16⁺) monocytes are depicted in the upper left quadrant, upper right quadrant and lower right quadrant, respectively. B) Principal component analyses (PCA) of single-cell PCR gene expression analysis data showing genetic clustering of the three monocyte subsets. The PCA plot confirmed the classification of the three human monocyte subsets done by flow cytometry, visualized by gene families. Each dot represents a single cell. C) Heatmap of gene expression values for PCA showing hierarchical clustering of single-cell PCR gene expression data from the three human monocyte sub-populations. The analysis revealed cellular heterogeneity by distinct gene signatures. Red circles = classical monocytes (<i>n</i> = 94 cells), green triangles = intermediate monocytes (<i>n</i> = 92 cells), and blue pluses = non-classical monocytes (<i>n</i> = 80 cells).</p