Preferential inhibition of adaptive immune system dynamics by glucocorticoids in patients after acute surgical trauma

Abstract

Glucocorticoids (GC) are a controversial yet commonly used intervention in the clinical management of acute inflammatory conditions, including sepsis or traumatic injury. In the context of major trauma such as surgery, concerns have been raised regarding adverse effects from GC, thereby necessitating a better understanding of how GCs modulate the immune response. Here we report the results of a randomized controlled trial (NCT02542592) in which we employ a high-dimensional mass cytometry approach to characterize innate and adaptive cell signaling dynamics after a major surgery (primary outcome) in patients treated with placebo or methylprednisolone (MP). A robust, unsupervised bootstrap clustering of immune cell subsets coupled with random forest analysis shows profound (AUC = 0.92, p-value = 3.16E-8) MP-induced alterations of immune cell signaling trajectories, particularly in the adaptive compartments. By contrast, key innate signaling responses previously associated with pain and functional recovery after surgery, including STAT3 and CREB phosphorylation, are not affected by MP. These results imply cell-specific and pathway-specific effects of GCs, and also prompt future studies to examine GCs' effects on clinical outcomes likely dependent on functional adaptive immune responses.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1. Study workflow.

a In a double-blind study, patients were randomized to receive a single preoperative dose of 125 mg methylprednisolone (MP, n = 28 patients) or saline placebo (n = 30 patients). Peripheral blood and clinical outcomes data were collected prior to surgery (baseline) and at the indicated time points after surgery. After erythrocyte lysis, peripheral immune cells were barcoded, stained with cell-phenotyping and intracellular cell-signaling antibodies, and analyzed by mass cytometry. Unsupervised bootstrapped clustering of immune cell subsets followed by random forest analysis was performed to identify differential immune cell dynamics in MP vs. control groups. b A non-linear dimensional reduction algorithm (Isomap) showing individual patients’ immunological trajectories after surgery along the innate (X) and adaptive (Y) axes (MP in red, control in black). Snapshots are shown for the 1 h, 6 h, 24 h, 48 h and 2 wk time points (animated trajectories can be found in Supplementary Movie 1). Right panel. Overview of median trajectories for the MP and control groups are shown in red and black.

Fig. 2. A high-resolution atlas detailing peripheral immune cell alterations by MP after surgery.

a Immune cells were clustered based on the expression of all phenotypic markers using an unsupervised bootstrapped clustering algorithm. The clusters were projected into two dimensions and major immune cell compartments were identified based on phenotypic marker expression (contoured in orange/green for innate/adaptive immune compartments, respectively). b Univariate p-values (two-sided Wilcoxon Rank Sum Test) were computed for each cluster at each time point to quantify the difference in functional marker expression or cell frequency between samples in the control (n = 30 patients) and MP (n = 28 patients) groups. At each time point, clusters were colored by the best univariate p-value observed for cell frequency and functional marker expression. c A Random Forest model was trained to classify patients in the control or MP group at each timepoint based on cluster-derived cell frequency and intracellular signaling responses. The boxplot depicts the probability predicted by the Random Forest model that samples from patients in the control (gray) or MP (blue) group were allocated to the MP group. The model revealed that samples from placebo- or MP-treated patients were distinguishable at 1 h (AUC = 0.91, p = 1.03E−7), 6 h (AUC = 0.92, p = 3.16E−8), 24 h (AUC = 0.85, p = 3.81E−6), and 48 h (AUC = 0.76, p = 3.2E−3) after surgery (two-sided Wilcoxon rank-sum test, p-values calculated for each unique model). All boxplots show median values, interquartile range, whiskers of 1.5 times interquartile range.

Fig. 3. Alteration of innate and adaptive immune cell frequencies by MP.

a Immune cell atlas depicting differences in cell frequency between the MP and control groups at 1, 6 and 24 h after surgery (expressed as % of CD45+ mononuclear cells, with the exception of neutrophils, which are expressed as % of total live cells). Cell clusters are color-coded according to the directional differences between the control (n = 30 patients) and MP (n = 28 patients) group. Directional differences were computed using a two-sided Wilcoxon rank-sum test (sign(r) -log(p-value), blue/red indicating an increased/decreased frequency in the MP group; arrows point at cell clusters that differ most significantly between the patient groups). In cell clusters of the adaptive immune compartment (contoured in green), MP treatment resulted in decreased frequencies of CD4+Tmem at 1 and 6 h and CD4+Tnaïve cells at 6 h, but no significant changes in CD8+T or B cell frequencies. In cell clusters of the innate immune compartment (contoured in orange), MP treatment resulted in decreased frequencies of cMCs and M-MDSCs at 1 h, and ncMCs and mDCs at 1 and 6 h. In contrast, MP resulted in increased frequencies of CD56loCD16+NK cells at 1 h and mDCs at 24 h. b, c Box-plots depicting the frequency of manually gated immune cell subsets corroborating observations contained in the immune cell atlas. Immune cell subsets for which MP’s effect on adaptive (b) and innate (c) immune cell frequencies were most pronounced (CD4+ Tmem, CD4+ Tnaive, cMCs) are shown. Neutrophil frequencies (not included in the immune cell atlas) are also shown. Box plots for all manually gated immune cells are available in Supplementary Fig. 4. All boxplots show median values, interquartile range, whiskers of 1.5 times interquartile range. (Two-sided Wilcoxon rank-sum test, *p < 0.01, **p < 0.001, ***p < 0.0001). Exact p-values are available in Supplementary Table 2.

Fig. 4. Alteration of intracellular pSTAT3 responses by MP.

a Immune cell atlas depicting differences of the phospho-(p)STAT3 response (arcsinh ratio) between the control (n = 30 patients) and MP (n = 28 patients) group at 1, 6, and 24 h after surgery relative to the preoperative time point. Blue/red cluster colors indicate increased/decreased signaling in the MP group, respectively (two-sided Wilcoxon rank-sum test). Arrows point at cell clusters that differ most significantly between the patient groups. In cell clusters of the adaptive compartment (contoured in green), MP treatment resulted in a sustained attenuation of pSTAT3 responses in CD4+T cells (first in Tbet+CD4+T cells at 1 h, then in CD4+ Tnaive and CD4+ Tmem cells at 6 and 24 h. In contrast, in clusters of the innate compartment (contoured in orange) MP treatment resulted in no significant changes in the pSTAT3 signal in monocyte subsets (including cMCs, intMCs, and M-MDSCs) and in only a modest increase in the pSTAT3 signal in CD56loCD16+NK cells at 1 and 6 h and ncMCs at 24 h. b, c Box plots depict the pSTAT3 signal in manually gated immune cell subsets corroborating observations contained in the immune atlas. b MP’s attenuation of the pSTAT3 signal was most pronounced in CD4+ T cell subsets. c MP does not attenuate the pSTAT3 signal in neutrophils (signal is increased at 24 h) or cMCs. All boxplots show median values, interquartile range, whiskers of 1.5 times interquartile range (two-sided Wilcoxon rank-sum test, *p < 0.01, **p < 0.001, ***p < 0.0001). Exact p-values are available in Supplementary Table 2.

Fig. 5. Alteration of total IκBα by MP.

a Immune cell atlas depicting differences in total IκBα (arcsinh ratio) between the control (n = 30 patients) and MP (n = 28 patients) group at 1, 6, and 24 h after surgery. Blue/red cluster colors indicate increased/decreased signaling in the MP group, respectively (two-sided Wilcoxon rank-sum test). MP treatment resulted in increased total IκBα in both the adaptive (CD4+Tmem, CD4+Tnaive, CD8+Tnaive and CD8+Tmem, contoured in green) and the innate compartment (CD56loCD16+ NK cells, mDCs, cMCs, ncMCs, M-MDSCs and DCs, contoured in orange, which was most prominent at 6 and 24 h after surgery. b, c Box-plots depict total IκBα in manually gated immune cell subsets corroborating observations contained in the immune atlas. Select immune cell subsets for which MP’s effect on total IκBα was most pronounced (CD8+ Tnaive and CD8+ Tmem, cMCs, and neutrophils) are shown. All boxplots show median values, interquartile range, and whiskers of 1.5 times interquartile range. (Two-sided Wilcoxon rank-sum test, *p < 0.01, **p < 0.001, ***p < 0.0001). Exact p-values are available in Supplementary Table 2.

Fig. 6. Clinical recovery outcome measures between control and MP treatment.

All boxplots show median values, interquartile range, whiskers of 1.5 times interquartile range of clinical recovery parameters (a) pain, (b) fatigue and daily functioning, and (c) functional impairment of the operated hip over the course of 28 days after surgery between control (n = 30 patients) and MP (n = 28 patients) groups (two-sided Wilcoxon rank sum test). Pain and functional impairment of the hip were assessed with an adapted version of the Western Ontario and McMaster Universities Arthritis Index (pain 0 to 40 = none to worst; function 0 to 60 = no to most severe impairment). Postoperative fatigue and daily functioning were assessed with the Surgical Recovery Scale (17 to 100 = worst to none).