Peer review of: Androgens predispose males to monocyte-mediated immunopathology by inducing the (2020)

Reviewers' comments NCOMMS-19-26999 
 
Reviewer #1 (Amebic liver abscess, amebiasis) (Remarks to the Author): 
The  authors  address  one  of  the  biggest  mysteries  in  parasitology,  which  is  why  amebic  liver 
abscess is predominantly (80-90%) a disease of young adult males. A male predominance of 
infection is also seen in several other infections and diseases, making the work presented here of 
broader significance. Prior work in the murine model of amebic liver abscess demonstrated that 
liver damage in hepatic amebiasis is due to CCL2 recruited monocytes that produce TNF alpha. 
 
Here the authors demonstrate that classical monocytes from males express higher levels of CCR2 
and  secreted  higher  levels  of  CXCL1.  In  contrast  monocytes  from  females  stimulated  with 
androgens led to increased expression of CXCL1. Neutralization of CXCL1 in the mouse model 
decreased abscess size in the liver demonstrating a causal role of CXCL1. 
 
1)  In  Figure 1  is  there  a  reduction  in Ly6c hi  monocytes  in  the  liver  abscess from a  CXCL1-
neutralized mouse? 
2) Is the reduction in CCR2 expression in monocytes seen in females of functional significance 
(chemotaxis in a gradient of CCL2)? 
 
Reviewer #2 (Hormone/immune crosstalk) (Remarks to the Author): 
 
In this article, Sellau et. al. used human PBMCs primarily stimulated with the TLR4 ligand, LPS, 
and  a  mouse  model  of hepatic  amebiasis  to  understand  the  role of  monocytes  in  driving  the 
differences in immune responses and immunopathology between males and females. The authors 
show that in humans as well as in a mouse model of parasitic infection, male-derived classical 
monocytes have greater chemoattractant properties and secrete more pro-inflammatory cytokines 
than  female-derived  cells,  which  likely  impacts  sex  differences  in  disease  pathogenesis.  The 
authors go on to show that androgens play an important role in drive this response of monocytes 
following stimulation. My specific comments are outlined below. 
 
Major: 
 
1. There is poor connection between the experiments. There is no doubt that the authors have 
done a lot of work. Missing, however, is the logical progression of the studies, which combine 
mouse  parasitic  infections  to  examine  sex  differences, human PBMC  studies  to evaluate  sex 
differences, human cells in vitro and transgender in vivo studies to consider androgens, and the 
use of a parasite that likely does not exclusively signal through TLR4 in mouse studies combined 
with the use of LPS, which does signal through TLR4, in human studies. There are a lot of data 
that  suggest  interactions,  but  no  unification  across  studies.  This  might  be  improved  by 
reorganizing the results. For example, the human in vitro data could be presented first, with the 
transgender male study presented in separate section, and mouse study placed at the end to 
show functional significance of these cells and effects of androgens (see below) in a disease 
pathogenesis model. The abstract reflects this problem and reads as a list of unrelated findings 
from a lot of different studies. 

2. The direct evidence suggesting the role of androgens in hepatic amebiasis is missing and
results in this study not fitting the theme of this paper (even the title of the paper).
Hormonal manipulations such as inclusion of gonadectomized mice (absence/reduction of
testosterone) treated with testosterone or DHT would further help to strengthen the argument that
androgens predispose males to enhanced immunopathology by providing.
3. Differential use of testosterone versus DHT in human studies. In the studies using blood from
transgender males, these individuals were taking testosterone (Fig 4d-f), which can signal through
both the estrogen and androgen receptor. In contrast, the studies of PBMCs from ‘health’ donors
involved isolating and treating cells in vitro with DHT (Fig 4g-i), which signals exclusively through
the androgen receptor. The authors need to resolve this experimental inconsistency.


Minor: 
1. Detailed information is lacking in multiple places. Texts and the figure legends should explain 
in detail about the sex of the animal used (e.g. 1J, K) and the age of men and women enrolled in 
the study. 
 
Reviewer  #3 
 
Authors have examined role of androgens in monocyte responses to LPS and amebic antigens. 
Some  of  the  results  are  interesting  but  the  results  and  conclusions  are  problematic. 
 
1. Many of the figures contain only 2 (two) independent experiments and still parametric statistics 
are used (Student t test). Also the differences are very small in many figures (fig 1h, 2c, 3b, 3b, 
3e, 5b) that do not warrant the conclusions stated by the authors especially considering the few 
experiments. 
 
2. Data on expression of molecules related to leukocyte migration do not warrant conclusion about 
actual migration. Effects on migration must be studied directly in order to make conclusions about 
it. 
 
3.  It  is  unclear  why  the  authors  used  LPS  when  wanting  to  make  conclusions  about  amebic 
infection.  Also  the  LPS  protocols  vary  throughout  the  manuscript  without  explanation. 
 
4. There are a lot of conclusions and speculations that should be deleted from the result section.