Interleukin-6 inhibition in ST-elevation myocardial infarction: Immune cell profile in the randomised ASSAIL-MI trial

Figures

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  Figure 1. Downregulatory effects by tocilizumab treatment on neutrophils and neutrophil-lymphocyte ratio (NLR) following STEMI. Note, Panel A and C show only the placebo group to compare the pattern in STEMI patients with levels in stable angina pectoris. Panel a shows the level of circulating neutrophils in STEMI patients treated with PCI (only placebo) over time compared with patients with stable angina pectoris (SAP) (n = 20) as mean with SEM. Grey-shaded area shows normal range. ****p < 0.0001 versus SAP (One-way ANOVA with Dunnett's multiple comparisons test). Panel b shows a comparison of the effect of tocilizumab treatment at all time-points between the neutrophil counts in the tocilizumab arm and in the placebo arm as mean with SEM. ****p < 0.0001 comparing the two treatment groups (Mixed effect analyses with Bonferroni's multiple comparisons test). Panel c shows NLR in STEMI patients treated with PCI (only placebo) over time compared with patients with stable angina pectoris (SAP) (n = 20) as mean with SEM. ****p < 0.0001 and **p < 0.01 versus SAP (Dunnett's multiple comparisons test). Panel d shows a comparison of the effect of tocilizumab treatment at all time-points between the…

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  Figure 2. Correlation between neutrophil counts and myocardial salvage index (MSI) and maximum troponin T (TnT) levels in STEMI patients. Panel a and b shows correlation between absolute neutrophil counts at 24 h and MSI (a) and maximum TnT levels (b) in the tocilizumab (T) and the placebo (P) group. Panel c and d show correlation between percent neutrophil change from admission to 24 h and MSI (c) and maximum TnT levels (d) in the tocilizumab (T) and the placebo (P) group. Trend line is indicated for easier visualization. Correlations were calculated using Spearman's correlation coefficient (r). Placebo (n = 98): at hospitalisation (n = 93), 24 h (n = 98). Tocilizumab (n = 101): at hospitalisation (n = 98), 24 h (n = 99).

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  Figure 3. Comparison of gene expression in whole blood after 3-7 days between placebo and tocilizumab treated STEMI patients. Panel a shows the number of significantly differentially regulated genes (adjusted p-value < 0.05) at hospitalisation (placebo [p]: n = 18, tocilizumab [t]: n = 19), 3-7 days after hospitalisation (p: n = 19, t: n = 20) and after 6 months (p: n = 18, t: n = 20) between the two treatment arms. Panel b shows the pattern of differential regulation between placebo and tocilizumab treated patients of the two Reactome pathways “Immune system” (R-HSA-168256) and “Signal transduction” (R-HSA-162582), when including all genes with a p-value < 0.01. Dark blue is significantly downregulated in the tocilizumab-treated compared with the placebo-treated patients, while bright yellow is significantly upregulated in the same groups.

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  Figure 4. Gene expression imputation in neutrophils derived from whole blood analyses. Panel a shows the 10 most enriched pathways (Reactome) and Gene Ontology (GO) terms from analysis with the gene expression level of the 423 genes belonging to neutrophils that had more than 50 average counts and a p-value < 0.01 (unpaired t test) after the deconvolution analysis performed on RNA-sequencing results from whole blood by CIBERSORTx. Panel b shows a network plot for the four most relevant Reactome pathways and GO terms for signal transduction, and Panel c shows pathways relevant for neutrophil function and cell development. Blue shows lower gene level (log2 fold change), while red shows higher gene level in the tocilizumab group compared with placebo treated patients. Panel d shows GSEA analysis for genes related to the Reactome pathway “Neutrophil degranulation”, and Panel e shows the GSEA analysis for the GO term “MAPK cascade”.

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  Figure 5. B cells, T cells and NK cells following STEMI. Levels of (a) NK cells, (b) B cells (CD19), (c) CD4+ T cells and (d) CD8+ T cells in STEMI patients (only placebo) at hospitalisation (n = 30), 24 h (n = 31), 3-7 days (n = 29) and 6 months (n = 30) compared with stabile angina pectoris (SAP) patients (n = 20). Data are presented as mean with SEM. Grey-shaded area: normal range. *p < 0.05 versus SAP (One-way ANOVA with Dunnett's multiple comparisons test). Data are given as mean and SEM. Hospital admission was within 6 h after symptom onset. Panel e shows significantly DEGs (adjusted p-value < 0.05, average gene expression > 50) in T cells between the treatment groups at hospitalisation (placebo [p]: n = 10, tocilizumab [t]: n = 6), 24 h after hospitalisation (p: n = 8, t: n = 8), after 3-7 days (p: n = 8, t: n = 8) and after 6 months (p: n = 10, t: n = 9).

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  Figure 6. Effect of tocilizumab on numbers of NK cells, B (CD19) cells, CD4+ T cells and CD8+ T cells according to time since symptom onset. Panel a shows numbers of NK cells, B (CD19) cells, CD4+ T cells, and CD8+ T cells at admission (before drug administration and PCI) according to time since symptom onset (≤ 3 h, n = 40 and > 3 h n = 24) as mean with SEM. Panel b shows CD8+ T cells counts for STEMI patients in the placebo and tocilizumab group based on time since symptom onset as mean with SEM. *p < 0.05 (Unpaired t test between relevant groups). Data are given as mean and SEM. Placebo ≤ 3 h (admission: n = 18, 24 h: n = 18, 3-7 days: n = 17, 6 months: n = 18); placebo > 3 h (admission: n = 12, 24 h: n = 13, 3-7 days: n = 12, 6 months: n = 12); tocilizumab ≤ 3 h (admission: n = 22, 24 h: n = 21, 3-7 days: n = 25, 6 months: n = 23); tocilizumab > 3 h (admission: n = 12, 24 h: n = 12, 3-7 days: n = 12, 6 months: n = 12). Hospital admission was within 6 h after symptom onset.