Peer review of: Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during (2021)

Reviewers' comments: 

Reviewer #1 (T.gondii neuro immunology)(Remarks to the Author): 

Overview of Manuscript: The manuscript addresses the specific roles of microglia, in infections in 
the CNS, using the neurotrophic pathogen, Toxoplasma gondii, which establishes a chronic 
infection in the brain, as an experimental model. To identify the specific roles of microglia, the 
authors used microglia reporter mice, sorted microglia from infiltrating myeloid cells, and analyzed 
microglia and macrophages using RNA sequencing. Using this approach it was found that NF-kB 
and inflammatory cytokines were overly expressed in macrophages vs. microglia and that IL-1α 
was enriched in microglia while IL-1β was enriched in macrophages. To probe this initial finding 
further the authors used a combination of knockout mice and chemical inhibition. Major findings 
were: 1) mice lacking IL-IR and IL-1α but not IL-1β, have impaired parasite control and immune 
cell infiltration, 2) microglia release IL-1α via a gasdermin-D-dependent mechanism and 3) 
gasdermin-D and caspase 11 deficient mice show deficits in parasite control and immune cell 
infiltration. The authors conclude macrophages and microglia are differentially equipped to 
propagate inflammation and that in chronic T. gondii infection, microglia release IL-1α which 
promotes neuroinflammation and parasite control. 

The manuscript aim to define the specific role of microglia is of importance as the role of this 
resident immune cell in the CNS is not well understood. The main findings of this study that 
microglia and macrophages are different in their abilities to propagate inflammation in the brain 
and that microglia release of IL-1α contributes to control of the chronic T. gondii infection, 
contributes both to the understanding of immune responses to infections in the CNS in general as 
well as to the understanding of the intracerebral immune response against Toxoplasmosis and will 
be of interest to the research community in these fields. This is a well-conducted study using 
cleverly designed experimental approaches with appropriate experimental controls. It is generally 
well written with the exception of a few issues regarding referencing and an over generalization or 
lack of understanding of literature and previous work on immune responses to T. gondii in the CNS, 
as detailed below. 

Specific Comments: 

Introduction 
p. 3, 3rd line – the authors state ‘As the only resident immune cell, microglia’. However, 
astrocytes can also serve as a resident immune effector cell in the brain….’ Furthermore, as 
studies show astrocytes are an important resident immune effector cell controlling T. gondii in the 
brain, some mention of astrocytes in this first paragraph should be given for proper and complete 
context (see works by Hunter 2016, 2004; Drögemüller et al., 2008, and others). 

p. 3, 2nd paragragh, 3rd sentence: The authors state in reference to the immune response to T. 
gondii “T-cell derived IFN-γ is one essential element” and cite 2 references. The literature on the 
immune response to T. gondii is voluminous and this simple statement referring to IFN-γ, as ‘one’ 
essential element is misleading about the role of IFN-γ. IFN-γ is ‘the’ essential cytokine controlling 
T. gondii in the brain with its role well defined by work by Y. Suzuki and numerous others. 
Additionally there is a wealth of knowledge of the immune response to this parasite in the brain. 
The work on the importance of IFN-γ in control of T. gondii by Suzuki should be cited as well as a 
more complete summary of the large body of knowledge known of the intracerebral immune 
response to T. gondii. 

Again, later in this paragraph the authors state the role of microglia in the chronic infection has 
not been fully ‘elucidated’ which is true, but Suzuki has done significant work on microglia in the 
acute phase (2007, 2005) which should be cited and referenced for broader context of what is 
known about the role of microglia in Toxoplasmosis in the brain. As the work presented in this 
manuscript primarily addresses the role of microglia in the chronic phase of the immune response 

to T. gondii and that aspect could be stated in the above statement to discriminate from what is 
known of microglia in the acute infection. 

Results 
p. 5, 1st paragraph – The statement “Blood-derived monocytes have been demonstrated to be 
important for host survival during infection, but whether microglia perform similar functions is still 
unknown” seems to be an overstatement and in part wrong. For example Suzuki’s work on 
microglia has shown that microglia are important as sentinel cells, able to detect early cerebral 
Toxoplasma proliferation and activate their IFN-γ production thus facilitating protection immunity 
in controlling parasite proliferation in the brain. This is essentially the same issue as noted above 
in the Introduction. 

Discussion 
p. 21 – 1st paragraph – The statement ‘Much is known about the immune response to this parasite 
but the role of the brain-resident microglia is still largely unknown.’ again reads as a little untrue 
or at least too broadly stated as the role of microglia in IFN-γ mediated protection as mentioned 
above is well established, and more recent evidence indicating migratory properties of infected 
microglia possibly serve to disseminate the parasite in the brain, are also in the literature. Hence 
this statement is just too general - a qualification of ‘in the chronic infection’ or some type of 
delineating/restricting condition, could be used to help clarify what is new in this manuscript while 
also indicating what is known of the understanding of microglia in Toxoplasmosis. 

Later in this paragraph the authors discuss the in vitro experiments done with T. gondii infected 
microglia and discuss how in vitro systems don’t adequately reflect the biology/phenotype of 
microglia in vivo. These in vitro studies are quite old and since that time most studies addressing 
the role of microglia have been done in vivo so this statement seems to ignore or at least 
erroneously suggest the in vitro studies are the main source of information on microglia and 
Toxoplasma. 

As a concluding statement to the authors, I wish to express that despite the above comments, I 
believe this is a well-done study is well-done that addresses an important question about the 
differential roles of macrophages and microglia in the brain in T. gondii which hasn’t received 
attention before. However the seeming lack of knowledge and/or understanding of the literature 
on work done on the role of microglia in the cerebral immune response to T. gondii bothered me 
throughout the manuscript. Nearly, all the specific comments refer to this issue, which occurred in 
several sections of the manuscript. However, addressing this issue I believe, will made this a 
better paper but also will put a greater emphasis on that in the manuscript that is truly new and 
noteworthy, and thus significantly improve the manuscript. 

Reviewer #2 (T.gondii Immune responses)(Remarks to the Author): 

Batisda et al follow a logical approach to delineate a molecular function for microglia in control of 
chronic T. gondii infection in the brain. They discover that rather than the expected NFkB 
transcriptional signature present in infiltrating macrophages, brain microglia are skewed to express 
IL-1a. These findings are corroborated on protein level. Mice lacking IL-1R1 and IL-1a, but not IL-
1b, exhibit impaired parasite control and immune cell infiltration into the brain in the chronic phase. 
Microglia ex vivo are shown to produce IL-1a, dependent on gasdermin D. Gasdermin D and 
caspase 1/11 knockout mice equally present with decreased parasite control and defect immune 
cell infiltration into the brain. 

This study addresses important differences between macrophages and microglia in controlling 
chronic T. gondii by profiling their inflammatory potential in the brain. IL-1a is the “neglected” IL-1 
in the context of inflammasome activation and IL-1R signalling, and this study adds to our 

knowledge of the different sources of these molecules. While the story is complete in what it set 
out to demonstrate, I have a few conceptual and technical questions. 

Questions: 
Microglia express IL-1a in the brain of T. gondii infected mice. IL-1a signals via IL-1R on 
endothelial cells to upregulate VCAM-1 and ICAM-1, which in turn recruit microglia to the brain. 
Chicken and egg question: Which comes first, IL-1a production in the brain by another cell type 
and then the amplification loop of IL-1a driven by recruited microglia or microglia recruited 
independently of IL-1a signalling, which then produce IL-1a leading to more microglia in the brain? 
In essence is there IL-1a production in the brain without microglia? 

What about IL-18, do the authors see any production of IL-18 in their system? 

The authors need to better discuss their findings in light of previously published work analysing the 
importance of casp1/11 and IL-1R during the acute phase of T. gondii infection (Gorfu et al). In 
the published study, these mice (esp. casp1/11KO) mostly do not make it to the chronic phase. I 
know the parasite stage used for infection is different, is that the reason why there is an observed 
difference in susceptibility? On the same note, there are >2000 cysts in the brains of their 
casp1/11KO mice – how do the mice survive that? Are they exhibiting signs of disease at that 
point? 

IL-1a processing is not dependent on inflammatory caspases. By studying gasderminD KO mice, 
the authors find that IL-1a release is dependent on inflammasome activation/pore formation. 
However, they do not analyse IL-1a levels in casp1/11KO mice. Presumably these would also be 
lower as casp1/11 processes gasdermin to form the pore? Is this the case? 

Minor comments: 
Figure S1b: + missing next to IL-1 
Figure 1: In the IF images, why is CX3CR1 labelled differently between panels e and f versus j and 
l? 
Figure 2 onwards in paper: Why are some experiments shown as paired averages comparing 
groups and others presumably are single points per mouse pooled from all experiments? 
P14 top: “cells” missing from sentence 
P18: citation for Gorfu et al is missing