Background
Transplantation, inflammatory, and auto-immune diseases have benefitted from the immunomodulatory properties of glucocorticoids for decades. Glucocorticoids modulate both innate and adaptive immune responses [
1]. They promote bone marrow release and survival of neutrophils. Glucocorticoids also modulate the innate response by suppressing pro-inflammatory or by stimulating anti-inflammatory mediators [
2]. Such balanced action promotes resolution of inflammation and prevents overshooting of the host response. This may contribute to the efficacy of dexamethasone in preventing morbidity and mortality in pneumococcal meningitis in children [
3]. Moreover, decreasing antigen presentation and co-stimulation by dendritic cells [
4] prevents the crosstalk between innate and adaptive systems. Glucocorticoids also promote a shift from T helper (Th)1 to Th2 cells, leading to impaired defense against intracellular and opportunistic infections [
5,
6].
The use of glucocorticoids in septic shock is an issue of incredible debate [
7]. Considering the aforementioned effects, it seems logical that glucocorticoids may avoid the deadly effect of the massive inflammatory response initially seen in sepsis. However, after acknowledging that high doses of glucocorticoids do not decrease mortality and may even be harmful [
8,
9], low (but still supra-physiologic) doses of hydrocortisone were assessed. While the reduction in mortality [
10] is still a matter of debate [
11,
12], hydrocortisone remains recommended for patients with refractory shock [
13,
14]. Indeed, hydrocortisone has almost always been associated with reversal of shock. The beneficial effect of corticosteroids on hemodynamics is highly intertwined with their effects on the inflammatory response and endothelium [
15]. Although the precise molecular mechanisms are still largely unknown, nitric oxide (NO) synthesis seems to play a determinant role in modulating the vascular tone over the initial course after injury [
16,
17].
For glucocorticoids, as for any other therapies evaluated for use in septic shock, the high heterogeneity of patients and the absence of stratification may explain inconclusive results. Given the amount of evidence supporting sepsis-induced immunosuppression [
18], the blind use of hydrocortisone - i.e. without monitoring the patient’s host response - may be questioned. Indeed, a retrospective study in pediatrics suggested that septic shock patients treated with glucocorticoids had numerous alterations of adaptive immunity at the transcriptional level [
19].
Severe burns and septic shock host responses share numerous features, including a relative late-stage state of immunosuppression. We recently described in a prospective, randomized, double-blind study that the use of hydrocortisone in refractory burn shock led to a statistically significant reduction in the duration of shock [
20]. As the impact of low-dose hydrocortisone on the host response has never been studied in patients with burns, we took advantage of this study to assess the whole blood transcriptional modulation in severe burn shock. Here, we studied the modulation of the immune response induced by shock and assessed the specific effects of hydrocortisone administration. We provide evidence that hydrocortisone transcriptionally enhances the immunosuppressive mechanisms that take place after severe injury.
Discussion
Here, we took advantage of a prospective, randomized, double-blind study to assess whole blood transcriptional modulation in severe burn shock and according to hydrocortisone administration. We identified wide and persistent modulation of gene expression over the first week after shock, whereas hydrocortisone-associated transcriptional modulation was moderate and transient. We also characterized the impact of both shock and hydrocortisone on the immune response, and showed that hydrocortisone transcriptionally enhanced the immunosuppressive mechanisms that occur after severe burn injury.
Few studies have evaluated the transcriptional host response to burn injury in humans. Modulation of gene expression after burn in skeletal muscle [
32,
33], skin [
34,
35], and adipose tissue [
36] leads to identification of pathways involved in metabolism, cell proliferation and inflammatory response, and osteogenic differentiation of mesenchymal cells, respectively. Regarding the blood transcriptome of burn patients, a first dataset (GSE19743, buffy-coat samples) from 57 patients and 63 healthy volunteers (analyzed in [
37‐
39]) showed that genes modulated within 10 days after injury were mainly related to immunity. Genes modulated at a later stage (11–49 days) were also involved in metabolism and apoptosis [
39]. Our results were consistent regarding the identified functional pathways. We showed that modulation was triggered early after burns as most genes were already modulated within 72 h of injury. A second dataset described the pooled-leukocytes transcriptome of 112 burn patients sampled within 7 days [
40]. This study assessed prognostic factors and found a 39-gene signature associated with a burn size >40%. These genes were related to platelet activation, TNF production, cellular adhesion, migration, and degranulation. Interestingly, our dataset shared 8 out of the 10 most modulated genes (
LCN2, LTF, THBS1, ITGA2B, CD24, TCN1, BPI and
SLC51A). Finally, we observed that most of the differentially expressed genes exhibited sustained modulation over time after burn injury. This is similar to a previous observation by Seok et al., whereby burn patients exhibited the longest period of transcriptome “recovery time” [
41].
The beneficial effect of hydrocortisone on the duration of septic shock is now widely accepted. We recently demonstrated a similar effect in burns, i.e. non-infectious/inflammatory shock [
20]. Glucocorticoids play important roles in the modulation of vascular tone. Although glucocorticoid-induced hypertension is primarily due to sodium retention and volume expansion, an increase in peripheral vascular resistance may also play a role [
42]. Here, we found no difference in adrenergic receptor expression in hydrocortisone-treated vs. placebo-treated patients. Several limitations might explain this negative result. First, the effects of hydrocortisone might be tissue-specific and not seen in the whole blood transcriptome. Second, the GR is known to have both genomic and non-genomic effects [
43], the latter being involved in the density of adrenergic receptors in vessels [
44] and the modulation of agonist-induced contractions in vascular smooth muscle at multiple sites along signal transduction pathways.
Glucocorticoid receptor may modulate other pathways involved in vascular tone such as NO. Indeed, we observed that the NO-mediated signal transduction pathway was up-modulated early after burn, but returned to control values quicker in hydrocortisone-treated than in placebo-treated patients. This result is consistent with former literature, as mice lacking endothelial GR were found to have higher levels of NO, and increased hemodynamic instability [
15]. Moreover, hydrocortisone administration to patients with septic shock was associated with reduction in plasma nitrite/nitrate (indicative of lower NO formation) and of vasopressor support [
17]. Taken together, these results are in favor of a hydrocortisone-induced modulation of NO balance that may explain positive hemodynamic effects in shock patients.
Glucocorticoids also have many side effects such as hyperglycemia, critical illness polyneuromyopathy [
45], delayed wound healing, and immunosuppression. These side effects and the absence of convincing results for the effect on mortality may explain the wide heterogeneity of practice in hydrocortisone administration in septic shock. Wong et al. have recently shown in pediatric patients with septic shock that corticosteroid administration was associated with greater repression of adaptive immunity-related genes [
19]. Despite well-matched groups in terms of severity, this study was retrospective, with no control over sampling time. Here, we confirmed the impact of hydrocortisone administration on host immune response in a different, but close model of inflammatory shock. Moreover, our prospective and randomized design allowed us to follow the hydrocortisone-related modulation of gene expression over a week.
Interestingly, along with greater repression of adaptive immunity, we also observed an impact of hydrocortisone on innate immunity. Indeed, the down-modulation of the antigen receptor-mediated pathway (Fig.
4b-c) was significantly greater at day 7 in the hydrocortisone group. This result was reminiscent of repressed monocyte expression of HLA-DR seen in various acute inflammatory responses, including burns, where it was associated with the occurrence of secondary septic shock [
46]. These results underline that hydrocortisone administration may deepen the immunosuppression associated with severe injury.
Interestingly, other groups have reported beneficial effects of hydrocortisone administration in injury-related models. In severe trauma, the incidence of hospital-acquired pneumonia was lower in the hydrocortisone group [
47]. The author’s hypothesis was that early hydrocortisone administration could blunt the hyper-inflammatory response associated with trauma, and prevent the subsequent associated immunosuppression. However, these results were not confirmed in a second multicenter trial published recently [
48]. In combination with our current results, this raises the question of: (1) the timing of hydrocortisone administration after injury, and (2) the duration of hydrocortisone administration. This also underlines the lack of tools to identify/stratify patients who may benefit from hydrocortisone.
Our study has several limitations. Despite an adequate design, the small sample size precluded us from assessing associations between hydrocortisone, host-response and outcomes such as mortality or secondary infections. As we selected only patients with severe shock (with >0.5 μg/kg/min norepinephrine), most of them had extensive burns (median TBSA = 70% (48–84), Table
1) and we found no transcriptional modulation according to TBSA. Therefore, we cannot extrapolate to the host response modulation in every patient with burns. However, this provided us with a very homogeneous cohort of patients, allowing us to more precisely decipher the pathways modulated after severe burn injury, and to identify similarities with inflammatory situations such as trauma and septic shock [
49]. As described in Table
1, several confounding factors might have impacted the transcriptome modulation over time (ABSI, etomidate administration, etc.). We observed no significant difference in the results when adjusting or not adjusting with these variables but the small sample size precludes a definitive conclusion. As all patients received blood transfusion during graft surgery, the impact of transfusion on transcriptome modulation could not be assessed. This deserves more specific evaluation in the future. Moreover, as we did not collect whole blood cell counts except at admission, we were not able to verify if changes in the pattern of blood leukocytes may have impacted longitudinal gene expression. Surprisingly, hydrocortisone treatment was only associated with a few modulated genes. Our small sample size and stringent thresholds for probe set filtering might explain such results. An additional explanation could be related to the profound basal modulation induced by burn injury, which might limit our ability to detect all hydrocortisone-modulated genes. However, such a design also allowed us to describe the impact of hydrocortisone on gene expression in vivo in an acute inflammatory situation, for the first time. Finally, our data were limited to mRNA expression. We were not able to test correlation with either translational modulation, or functionality of the immune system. Demonstration of altered immune functionality in burn patients is thus still pending.
Acknowledgements
We thank Dr Marc Bertin-Maghit, Dr Christophe Magnin, and Dr Laure Fayolle-Pivot, for their help in recruiting the patients in the prospective study. This project is part of Advanced Diagnostic for New Therapeutic Approaches (ADNA), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by the French public agency BPI France.