Peer review of: Preferential inhibition of adaptive immune system dynamics by glucocorticoids in patients (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
In this study, the group used a cohort of surgical patients to examine the dynamics of how surgical 
trauma alters the circulating immune cell subset landscape with a comparison of the effect of 
methylprednisolone (MP) treatment on immune cell phenotypes. They used 11 phospho-signaling 
mediators as functional cellular readouts for how MP changes innate and adaptive immune cell subsets. 
The study uses an appropriate and well thought out data analysis workflow to reduce the complex 
high-dimensional single cell analysis by mass cytometry into interpretable results. In particular, the 
random-forest algorithm worked well to demonstrate that the majority of significant changes in 
cellular phenotypes occurred within 48 hours after surgical trauma. There are several new 
contributions to the trauma immunology field that have basic science and clinical implications. First, 
MP had the most significant effect on cytokine signaling responses by adaptive immune cell types and 
did not markedly change innate cells (except for IkBa): Second, most of the highly significant 
circulatory immune changes to trauma occurs within 3 days after the insult. This is consistent with 
already published work for surgical trauma and in trauma patients. 
The mass cytometry data analysis is a strength of the study. Another strength is that the MP 
treatment had a significant effect on specific signaling pathways in adaptive immune cell types. The 
work will be of general interest to the surgery and trauma field and does contribute novel information. 
There are some issues that should be addressed for clarity and to increase the impact of the study: 
1. The format of the cell clusters in 2-dimensional space is somewhat hard to see, especially with the 
signaling studies. 
2. Along this same issue, how did they generate these cluster plots? Were they down sampled and 
how many events were used for the clusters. Also, are the frequencies relative or normalized to be 
similar as event dots? These 2-dimensional plots are not what is normally seen in mass cytometry or 
single-cell analysis studies, so clarifying how these were generated and associated information should 
be included in the manuscript. 
3. The clinical finding was that MP treatment had no measurable effect on recovery or post-operative 
pain conditions. The discussion section suggests that giving MP treatment to patients after surgery 
may not be a good idea given that the treatment inhibits adaptive immune cells and that might have 
detrimental effects on host immune function – in particular, post-operative infections. Did the authors 
examine post-operative infection rates in the control and MP treated patients? This should be indicated 
in the manuscript text or added as a clinical outcome. 
4. What positive controls do the authors use to validate their signaling antibody stains with the smart 
tube approach that they are using? 
5. In Supplemental Figure 2, it is difficult to see the arrows, please increase their size or make the 
arrow ends bigger 
6. The CD7 marker plot in the gating scheme is confusing – it indicates NK cells that are CD7+/CD14-. 
Is this an error in the figure? Usually CD56 or CD94 is used on CD3- cells to delineate NK cells? This 
review thinks that CD7 is on T cells, but could be wrong. 
7. Supplemental Figure 4 is unreadable. The authors need to figure out a better way to present these 
data. This is challenging but needed, the figure cannot be enlarged with good resolution either. 
 
James Lederer 
 
 
Reviewer #2 (Remarks to the Author): 
 
In this manuscript, the authors describe a mass cytometry approach to study changes in the immune 
cell landscape and relevant signal transduction pathways after a single dose of glucocorticoids (GCs) 

applied to patients before surgery for total hip replacement. In particular, the authors analyzed the 
abundance of a variety of innate and adaptive immune cell subtypes in the blood and characterized 
the activity of the JAK/STAT, NFkB and MAPK pathways at several time points after surgery. The 
experiments are technically sound and the work provides a comprehensive overview of the changes 
induced by GCs in immune cells. However, the study has also its limitations and suffers from the fact 
that the observed alterations were not linked to differences in clinical outcome. There are a number of 
concerns related to this study which need to be addressed and/or discussed. 
 
1. The study includes only patients of 55-80 years of age, which is of course intrinsic to the study 
design since very few young individuals will undergo a hip replacement. Nevertheless, this feature 
brings about that many of the test persons presumably have an impaired immune system due to 
immunosenescence. Therefore it is unclear to which extent the findings will reflect the situation of a 
fully functional immune system present in healthy young individuals. Rather, some of the observations 
may be related to the decreased activity and abundance of specific immune cell subsets in elder 
individuals. 
 
2. Supplementary Figure 4 is much too small and should be subdivided into several separate figures. 
Currently, it is impossible to read and interpret the data contained therein. 
 
3. It is noteworthy that several findings described in this manuscript are trivial and have been known 
for a long time. For instance, the observation that CD4+ T cells rapidly decrease after MP treatment 
simply reflect the high sensitivity to apoptosis induction of this cell type, a process that has been 
studied for decades. Similarly, the fact that the number of neutrophils rapidly increases after MP 
injection is also well known and is a consequence of demargination mediated by altered expression of 
adhesion molecules. 
 
4. Inherent to the analytical method, the reported results focus on the abundance of immune cells in 
the blood and the activity of signal transduction pathways. No information, however, is provided on 
processes such as proliferation, apoptosis, migration and adhesion. Thus, the mechanistic insights are 
limited although the information is still important. 
 
5. An interesting finding is that MP inhibits Stat3/5 phosphorylation in T cells but almost not in 
neutrophils and monocytes. In contrast, IkBa is upregulated in both innate and adaptive immune cells. 
However, it is possible that this and other observations are related to the immunosenescence in the 
analyzed cohort of elder patients rather than being a general phenomenon. It would thus be important 
to validate some of the data in a cohort of younger patients with a different diagnosis but also treated 
with MP to check whether similar changes apply to individuals with a fully functional immune system. 
 
6. The authors should comment on whether the single dose of MP had any effect on post-surgery 
vomiting and nausea compared to the placebo group. Of note, this effect of GCs is not linked to their 
anti-inflammatory activity as suggested by the authors in the introduction. 
 
7. Could it be that the lack of efficacy of MP on the clinical outcome, namely pain and functional 
recovery 4 weeks after surgery, is due to the fact that GCs were only injected once? Would the 
outcome be different if GCs were repeatedly administered instead? This issue should be discussed in 
more detail. 
 
8. The induction of IkBa is only one out of several mechanisms by which GCs inhibit NFkB signaling. 
As a matter of fact, interaction of the GC receptor with NFkB in the nucleus via protein-protein-
interaction is believed to be the more relevant mechanism in this context (e.g. reviewed in Nat Rev 
Mol Cell Biol 2017, 18:159-174). Such an effect, however, cannot be addressed by the approach used 

in this manuscript. Interpretations concerning the impact of GCs on NFkB signaling should thus be 
made with caution.